Glycopeptides derived from pancreatic structures, antibodies and applications thereof in diagnostics and therapeutics

ABSTRACT

Glycopeptide comprising 1 to 40 repeated C-terminal polypeptides, with 11 amino acids, of BSDL or FAPP, whereby said polypeptides are glycosylated and bear glycosylated epitopes giving rise to a specific immunological reaction with induced antibodies in a patient suffering from type 1 diabetes and/or purified from biological fluids of human or animal origin or recombinant and produced by expression in a standard host cell comprising an enzymatic material necessary for priming a glycosylation, said host cell being genetically modified such as to comprise a gene coding for said polypeptides and a gene coding for one or more enzymes selected from among glycosyltransferases, anti-glycopeptide antibodies and the applications thereof in therapeutics and diagnostics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/593,859, filed Sep.22, 2006, which is the U.S. national stage application of InternationalPatent Application No. PCT/FR2005/000771, filed Mar. 30, 2005, thedisclosures of which are hereby incorporated by reference in theirentirety, including all figures, tables and amino acid or nucleic acidsequences.

The invention relates to glycopeptides derived from pancreaticstructures, antibodies and applications thereof in diagnostics andtherapeutics.

More particularly, the invention relates to the obtaining of naturalC-terminal glycopeptides of BSDL and/or FAPP carrying variousglycosylated epitopes (alone or combined) recognized by inducedantibodies in a patient suffering from type I diabetes and purified frombiological fluids of human or animal origin as well as to saidrecombinant glycopeptides produced by biological engineering from celllines containing cDNAs coding for the C-terminal peptides of FAPP and/orBSDL and cDNAs coding for the different glycosyltransferases required tobuild said glycopeptides. The invention also relates to monoclonalantibodies recognizing the recombinant and/or natural C-terminalglycopeptides of BSDL and/or FAPP, and to the use of said glycopeptidesand said monoclonal antibodies as immunogenic agents, diagnostic agentsand therapeutic agents that can be used for the diagnosis or thepreventive or curative treatment of pancreatic pathologies such asdiabetes and cancer of the exocrine pancreas or any other pathologies aswell as in curative or preventive protocols of said pathologies.

Cancer of the exocrine pancreas, which accounts for over 20% ofdigestive tract cancers, is one of the most aggressive. In France forexample, 4,000 new cases are diagnosed each year. Furthermore, itsfrequency is rising markedly in many regions of the world. Survivalrates do not exceed 20% at 1 year and 3% at 5 years and mean survival is3 to 4 months after diagnosis. The cancer is diagnosed late andprogresses very rapidly, mainly through the formation of peritoneal andhepatic metastases. In addition, the deep anatomic location of thetumor, the absence of sensitive and specific early biological markersand its asymptomatic nature result in a diagnosis that always occurslate. Currently there are no effective therapies for exocrine pancreaticcancer, which is refractory to chemotherapy and radiation.

It would therefore be desirable to have specific markers of pancreaticcancer or other pancreatic pathologies available so as to diagnose thesediseases and have products to provide effective treatments.

Now, after extensive research, and in a surprising manner, theapplicants have discovered specific markers of pancreatic pathologies,and in particular of pancreatic cancer, expressed at the surface of thepancreatic tumor cell.

The applicants have discovered circulating antibodies directed againstglycosylated antigenic structures of the C-terminal peptides of bilesalt dependent lipase (BSDL), in diabetic patients.

The applicants have also discovered that monoclonal and polyclonalantibodies anti-glycoslated epitope expressed in the C-terminal part ofhuman fetoacinar pancreatic protein (FAPP) specifically recognized humanpancreatic tumor cell lines, but did not recognize tumor cell lineswhich were not of pancreatic origin.

The applicants have also discovered that monoclonal and polyclonalantibodies anti-glycosylated epitope expressed in the C-terminal part ofhuman fetoacinar pancreatic protein (FAPP) specifically recognized humanpancreatic tumor tissue, but did not recognize normal pancreatic tissue.

The applicants have further discovered that the antibodiesanti-glycosylated epitope expressed in the C-terminal part of BSDL orFAPP could detect BSDL- and FAPP-derived glycopeptides in urine. Inparticular, the antibodies anti-glycosylated epitope expressed in theC-terminal part of FAPP could detect glycopeptides in urine allowing toidentify a subject with a pancreatic pathology, particularly pancreaticcancer.

More particularly, therefore, the applicants have discovered thatcompounds having a glycopeptide structure whose peptide part is based onthe repeated C-terminal sequences of BSDL, a digestive lipolytic enzymepresent in normal pancreatic secretions, or based on the repeatedC-terminal sequences of FAPP (an oncofetal form of BSDL) constitutedsuch specific markers of pancreatic pathologies.

Indeed, BSDL and FAPP comprise repeated C-terminal peptide sequences of11 amino acids, comprising a generally invariant part with 7 amino acidshaving the sequence Ala Pro Pro Val Pro Pro Thr and a glycosylationsite. Said generally invariant part is flanked on either side by aglycine often substituted by a glutamic acid and contains the aminoacids Asp and Ser on the N-terminal side.

The applicants have also discovered that the compounds having aglycopeptide structure which could be prepared by expression andsecretion by a host cell, for example from Chinese hamster ovary (CHO),comprising a gene construct including a DNA molecule coding for one ormore repeated sequences of the C-terminal peptide, particularlyrecombinant of BSDL, for example all or part of the 16 repeatedsequences and also comprising a gene construct such as a DNA moleculecoding for at least one enzyme with ose-transferase activity, inparticular selected in the group consisting of Core 2 β(1-6)N-acetylglucosaminyltransferase, fucosyltransferase FUT3 which hasα(1-3) and α(1-4) fucosyltransferase activity, or fucosyltransferaseFUT7 which only has α(1-3) fucosyltransferase activity, constituted saidspecific markers of pancreatic cancer.

Thus the present application has as object a glycopeptide, particularlyrecombinant, possibly isolated or purified, comprising from 1 to 40repeated C-terminal polypeptides, composed of 11 amino acids, of BSDL orFAPP, said polypeptides being glycosylated and carrying glycosylatedepitopes giving rise to a specific immunological reaction with inducedantibodies in a patient with type I diabetes and

-   -   or else purified from biological fluids of human or animal        origin    -   or else recombinant and being produced by expression in a        conventional host cell comprising an enzymatic machinery        necessary for priming a glycosylation, said host cell being        genetically modified so as to comprise a gene coding for said        polypeptide and a gene coding for one or more enzymes selected        from glycosyltransferases and in particular from Core2 β(1-6)        N-acetylglucosaminyltransferase (abbreviated C2GnT), α(1-3)        galactosyltransferase, fucosyltransferase 3 (abbreviated FUT3)        and fucosyltransferase 7 (abbreviated FUT7).

In other preferred conditions of implementation of the invention, theaforementioned glycopeptide is essentially constituted of 1 to 40glycosylated repeated C-terminal peptides, with 11 amino acids of BSDLor FAPP, and in particular exclusively constituted of said glycosylatedpeptides. Hence, the invention therefore relates to isolated, purifiedor recombinant glycopeptides comprising, or essentially consisting of,repetitions of the repeated C-terminal peptide sequence of 11 aminoacids, preferably: D-S-G/E-A-P-P-V-P-P-T-G/E (SEQ ID No 14). Saidglycopeptides can contain 1 to 40 repetitions. In a preferredembodiment, said glycopeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 repetitions.Preferably, the glycopeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 repetitions. In aparticular embodiment, the glycopeptides comprise 1, 2, 3, 4, 5, 7, 8,9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 repetitions. Inanother alternative embodiment, the glycopeptides comprise between 1 and15 repetitions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 repetitions), preferably between 2 and 10 repetitions (forexample, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repetitions). In an additionalembodiment, the glycopeptides comprise between 17 and 40 repetitions(for example, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 repetitions). In preferredconditions of carrying out the invention, the aforementionedglycopeptide comprises from 1 to 40, preferably from 4 to 25 and moreparticularly from 6 to 16 repeated C-terminal polypeptides. In aparticular embodiment, the glycopeptide comprises, or essentiallyconsists of, 6 repetitions.

Preferably, the glycopeptides according to the invention areglycosylated by one or more enzymes having ose-transferase activityselected in the group consisting of Core 2 β(1-6)N-acetylglucosaminyltransferase (C2GnT), fucosyltransferase FUT3 whichhas α (1-3) and α(1-4) fucosyltransferase activity, orfucosyltransferase FUT7 which only has α(1-3) fucosyltransferaseactivity. In a particular embodiment, the glycopeptides according to theinvention have been glycosylated by the enzymes C2GnT and FUT3. In analternative embodiment, the glycopeptides according to the inventionhave been glycosylated by the enzymes C2GnT and FUT7. The invention alsoencompasses glycopeptides which have been glycosylated by the enzymesC2GnT, FUT3 and FUT7. In a preferred embodiment, the glycopeptidesaccording to the invention have additionally been glycosylated byα(1-3)galactosyltransferase (GT). Thus, when the glycopeptide accordingto the invention is recombinant, the host cell which produces itcomprises a gene coding for said polypeptide and a gene coding for oneor more enzymes selected from glycosyltransferases and in particularfrom among C2GnT, FUT3 and FUT7. In a preferred embodiment, said hostcell additionally comprises a gene coding forα(1-3)galactosyltransferase (GT).

In a particularly preferred embodiment, the invention relates to arecombinant, isolated or purified glycopeptide comprising 1 to 40repetitions of the peptide sequence described in SEQ ID No 14 andglycosylated by one or more enzymes having ose-transferase activityselected in the group consisting of Core 2 β(1-6)N-acetylglucosaminyltransferase (C2GnT), fucosyltransferase FUT3 whichhas α (1-3) and α(1-4) fucosyltransferase activity, orfucosyltransferase FUT7 which has α(1-3) fucosyltransferase activity,said glycopeptide additionally being glycosylated byα(1-3)galactosyltransferase (GT).

“Isolated glycopeptides” is understood to mean that said compounds areseparated from their natural environment. Thus, said compounds areseparated from some or all of the other components of their naturalenvironment.

“Purified glycopeptides” is understood to mean that said compounds areenriched in a mixture by at least approximately a factor of 1,preferably by at least 2, 3, 4 or 5-fold. The term “purified” does notnecessarily mean that the compounds are absolutely pure. For instance,said compounds can have a purity of 30%, 40%, 50%, 60%, 70%, 80%, 90% or100%. Preferably, however, said compounds generate only single bands inpolyacrylamide gel electrophoresis.

Any suitable separation and/or purification method by which to obtainthem can be used. In a particular embodiment, the glycopeptides arepurified from a biological fluid from a human or animal subject. Thebiological fluid can be selected in the group consisting of serum,urine, pancreatic juices and milk secretions. Preferably, the biologicalfluid is urine. In a preferred embodiment, the subject is a patient whomay be or who is suffering from a pancreatic disease, preferablypancreatic cancer. In another embodiment, the glycopeptides are purifiedfrom a cell culture. In an additional embodiment, the glycopeptides arepurified from a tissue sample, preferably pancreatic tissue and inparticular a pancreatic tissue tumor.

In the spirit of the application, the term “glycotype” refers toglycosylated epitopes. The glycosylated epitopes can involve one or moreglycosylated chains. The term “mAb” (“Acm” in French) refers to amonoclonal antibody, the term “pAb” (“Acp” in French) to a polyclonalantibody.

In a particular embodiment, the invention relates to a glycopeptideaccording to the invention which can give rise to a specificimmunological reaction with an antibody according to the invention.Preferably, the glycopeptide can give rise to a specific immunologicalreaction with one or more antibodies selected from J28 and 16D10.Optionally, the glycopeptide will give rise to a specific immunologicalreaction with J28. Alternatively, it will give rise to a specificimmunological reaction with 16D10.

The host cells classically used in genetic engineering naturally containenzymes, for example α-3 N-acetylgalactosaminyltransferase which adds anN-acetylgalactosamine generally on a serine or threonine residue of apeptide. For example this is the case for widely used host cells such asC127, COS, CHO or CHO-K1 which contain such enzymes.

In the present application and in that which follows, the term“conventional host cell” denotes a cell normally used for the productionof glycopeptides, preferably a human cell or an animal cell such asC127, COS cells, or insect cells such as Spodoptera frugiperda ovarycells and particularly a CHO or CHO-K1 cell. Such a suitableconventional host cell can be selected in particular on the basis of itsgenetic machinery and its abilities to generate various glycosylatedepitopes. To make said selection, an enzymatic assay can be carried outto detect the presence in particular of α-3N-acetylglucosaminyltransferase in the host cell that one wishes to use.

The glycosyltransferases which are used can be of animal or preferablyhuman origin.

In preferred conditions of implementation of the invention, thegenetically modified conventional host cell comprises at least one genecoding for said polypeptide and at least one gene coding for Core2β(1-6) N-acetylglucosaminyltransferase (C2GnT) and fucosyltransferase 3(FUT3).

In other preferred conditions of implementation of the invention,particularly for applications in pancreatic cancer, the geneticallymodified conventional host cell comprises at least one gene coding forsaid polypeptide and at least one gene coding for Core2 β(1-6)N-acetylglucosaminyltransferase (C2GnT), fucosyltransferase 3 (FUT3) andα (1-3)galactosyltransferase (GT).

In still other preferred conditions of implementation of the invention,particularly for applications in type I diabetes, the geneticallymodified conventional host cell comprises at least one gene coding forsaid polypeptide and at least one gene coding for glycosyltransferaseCore2 β(1-6) N-acetylglucosaminyltransferase (C2GnT) andfucosyltransferase 7 (FUT7).

All or part of the repeated C-terminal peptides can be glycosylated.Preferably, all of the repeated sequences will be glycosylated,generally 16 sequences, more specifically 6 sequences and in particular2 sequences will be glycosylated.

The glycosylations can be due to one or more N-acetylglucosamine,N-acetylgalactosamine, sialic acid, glucose, galactose, fucose.

In yet other preferred conditions of implementation of the invention,the size of an aforementioned glycopeptide estimated by polyacrylamidegel electrophoresis (SDS-PAGE) is advantageously comprised between 20and 120 kDa, preferably comprised between 20 and 85 kDa, moreparticularly comprised between 45 and 83 kDa.

In particular, the glycopeptides named C2-F3-16R, C2-F3-GT-6R and moreparticularly C2-F3-6R and C2-F7-16R are considered. C2-F3-16R designatesa glycopeptide having 16 repetitions glycosylated by C2GnT and FUT3.C2-F3-GT-6R designates a glycopeptide having 6 repetitions glycosylatedby C2GnT, FUT3 and GT. C2-F3-6R designates a glycopeptide having 6repetitions glycosylated by C2GnT and FUT3. C2-F7-16R designates aglycopeptide having 16 repetitions glycosylated by C2GnT and FUT7.Preferably, the glycopeptides according to the invention are selected inthe group consisting of C2-F3-6R, C2-F3-GT-6R, C2-F7-6R, C2-F7-GT-6R,C2-F3-F7-6R, and C2-F3-F7-GT-6R.

A cell line producing the glycopeptide C2-F7-16R was deposited with theCollection Nationale de Culture de Microorganismes (CNCM), InstitutPasteur, 28, rue du Dr Roux, 75724 Paris Cédex 15, France on 16 Mar.2004 under the number I-3189.

The person skilled in the art knows that a peptide can undergo severalstructural modifications without altering function.

The peptide can contain one or more modifications in its amino acidssuch as deletions, substitutions, additions or functionalizations suchas acylation of amino acids, in so far as said modifications do notaffect the immunologic characteristics thereof. In particular, themodification or modifications can be substitutions by a conservativeamino acid (that is, having similar physicochemical characteristics).For example, in general, the substitution of a leucine residue by anisoleucine residue does not alter such properties. Generally, themodifications must concern less than 40%, in particular less than 30%,preferably less than 20% and more particularly less than 10% of theamino acids of the peptide. It is important that the modified peptidedoes not be denatured such as can be done for example by a physicaltreatment such as heat, so as to preserve the conformational sitesthereof so that the antibodies induced by the modified derivatives willbe active towards the native structure.

In general, with respect to the modifications, the homology orsimilarity between the aforementioned modified glycopeptide and theaforementioned native glycopeptide, as well as to the modes of use, orof coupling the immunogenic compound according to the invention to animmunogenic protein like tetanus toxoid, reference can be made inparticular to WO-A-86/06 414 or to EP-A-0.220.273 or else toPCT/US.86/00831, equivalent.

The glycopeptides which are the object of the invention can be preparedin particular from pancreatic juices, serum, milk, urine or amnioticfluid of human or animal origin. In a preferred embodiment, theglycopeptides according to the invention are isolated and/or purifiedfrom urine of human or animal origin. Preferably, the urine is of humanorigin. In a preferred embodiment, the urine is from a patient who maybe or who is suffering from a pancreatic pathology. Preferably, thepancreatic pathology is pancreatic cancer. Those skilled in the art arefamiliar with the techniques that can be used to purify and/or isolatethe inventive glycopeptides. In particular, the glycopeptides can bepurified by using for example, but without being limited to saidmethods, centrifugations, ultrafiltrations, concentration steps, liquidchromatography on exclusion or affinity columns, reverse phasechromatography or cation or anion exchange chromatography,electrophoresis or a combination of said techniques. In particular, theantibodies specific of the glycopeptides according to the invention suchas J28 and 16D10 can be used for an affinity purification. An example ofa purification protocol is described in Experiment 7. Thus, theinvention relates to a method of preparation of a glycopeptide accordingto the invention comprising collecting the urine of a subject andpurifying said glycopeptide from the urine.

The invention more particularly relates to glycopeptides according tothe invention which are purified and/or isolated from the urine of apatient who may be or who is suffering from a pancreatic pathology, inparticular pancreatic cancer. As described hereinbelow, said purifiedglycopeptides can be used for preventive or therapeutic treatment byimmunotherapy. For instance, they can be used to prepare apharmaceutical and/or vaccine composition.

The inventive glycopeptides can be recombinant and prepared as follows.

A suitable conventional host cell line like CHO-K1 is transfected with aplasmid comprising the complementary DNA of the desired transferase orby plasmids comprising the complementary DNAs of the desiredtransferases, for example the glycosyltransferase Core2 β(1-6)N-acetylglucosaminyltransferase (C2GnT), fucosyltransferase 3 (FUT3),fucosyltransferase 7 (FUT7) or α(1-3)galactosyltransferase (GT), byusing lipofectamine for example.

The resulting cell line is transfected with a plasmid enabling theexpression and preferably the secretion of the recombinant peptides, forexample a pSecTag plasmid containing the cDNA coding for an inventiveglycopeptide, for example the C-terminal domain of BSDL or FAPP. SEQ IDNos 6, 8, 10 and 12 are examples of sequences coding for a glycopeptideaccording to the invention.

The recombinant glycopeptide obtained by culturing the cell linecontaining the desired cDNAs is then advantageously purified for exampleon a polyacrylamide gel or by liquid and/or affinity chromatography andif desired it is analyzed by SDS-PAGE for identification.

Of course it is possible to transfer the genes required to prepare theinventive glycopeptides into the host cell by any other suitable method.

In preferred conditions of implementation of the aforementioned method,a suitable cell line like CHO-K1 is transfected with a plasmidcomprising the complementary DNA of glycosyltransferase Core2 β(1-6)N-acetylglucosaminyltransferase (C2GnT), fucosyltransferase 3 (FUT3),and a glycopeptide according to the invention, for example theC-terminal polypeptide domain of FAPP or in particular of BSDL,preferably in that order.

In an advantageous manner, the CHO cell line and in particular theCHO-K1 cell line is used as host cell.

In an advantageous manner, the plasmids pSecTag, pcDNA-3 or pBK-CMV areused as plasmid.

More generally, the invention also has as object a method of preparationof a glycopeptide such as described hereinabove, characterized in thatone transfers into a conventional host cell comprising an enzymaticmachinery necessary for priming a glycosylation, a gene coding for thepolypeptide according to the invention and at least one gene coding forone or more enzymes selected from glycosyltransferases, in conditionsenabling the expression and optionally the secretion of the desiredglycopeptide, and one isolates the expected glycopeptide.

The glycopeptides according to the invention, and in particular thenatural or derivative glycopeptides of FAPP or BSDL, can be preparedfrom proteins isolated from biological fluids or tissues of any human oranimal origin whatsoever, particularly sera, urine, pancreatic juices,milk secretions, tissue or cell homogenates, and the like, bychromatographic methods such as described by Lombardo et al. 1978,Biochim. Biophys. Acta, 527: 142-149, or by Abouakil et al. 1988,Biochim. Biophys. Acta, 961: 299-308, or by Wang & Johnson, 1983, Anal.Biochem., 133: 457-461 or else by Blackberg & Hernell, 1981, Eur J.Biochem. 116: 221-225. The homogeneous proteins are then hydrolyzedeither by enzymatic methods involving any enzyme particularlyproteolytic, or by chemical methods.

The natural glycopeptides can be isolated by chromatographic methods(gel filtration, affinity and immunoaffinity, ion exchange, etc.) asdescribed in particular in Mas et al. 1997, Glycobiology, 7: 745-752, orWang et al. 1995, Biochemistry, 34: 10639-10644, or by any other method.

Said glycopeptides can also be purified, isolated either directly frombiological fluids or tissues as indicated earlier or subsequent to theaforementioned enzymatic or chemical treatments of said biologicalfluids or tissues.

The glycosylation of said natural glycopeptides of BSDL or FAPP can bemodified by any chemical or enzymatic routes (use of native, solublerecombinant, membranar in particular, of glycosidases orglycosyltransferases) in order to obtain the glycan structureshereinbelow used in particular for the diagnosis of the pathologiesindicated hereinabove and hereinbelow or for vaccination against saidpathologies, or for obtaining or producing antibodies which are theobject of the invention.

In addition, it is possible to modify the peptide sequence of thenatural or recombinant glycopeptides of the invention.

The natural and particularly recombinant, isolated or purifiedglycopeptides which are object of the invention have very interestingproperties. In particular, they exhibit remarkable immunogenicproperties, specific of pancreatic pathologies.

Said properties are illustrated hereinbelow in the experimental section.They justify the use of the aforementioned glycopeptides as medicament.

This is why the invention also has as object the aforementionedglycopeptides, for the use thereof in a method of therapeutic treatmentof the human or animal body, that is to say, as medicament.

The medicaments according to the invention can be used for example inimmunotherapy (immunization, vaccination) preventive and/or curativetreatment of pancreatic cancer, breast cancer, for cell-basedimmunotherapy: autologous vaccination by activation of the patient'simmune system (dendritic or other cells, etc.), for the diagnosis ofcertain pathologies such as type I diabetes by detection of circulatingantibodies directed against said structures in patients afflicted with adiabetic pathology and more generally with any other pathology requiringthe use of one or more of said glycopeptides.

They can also be used in other biological systems such as inflammation,the formation of metastases and the inhibition of invasion by pathogens.

Thus, the invention relates to a pharmaceutical or vaccine compositioncomprising a glycopeptide according to the invention. In particular, theinvention relates to a pharmaceutical or vaccine composition comprisinga glycopeptide comprising 1 to 40 repetitions of the peptide sequencedescribed in SEQ ID No 14 and glycosylated by one or more enzymes havingose-transferase activity selected in the group consisting of Core 2β(1-6) N-acetylglucosaminyltransferase (C2GnT), fucosyltransferase FUT3which has α(1-3) and α(1-4) fucosyltransferase activity, orfucosyltransferase FUT7 which has α(1-3) fucosyltransferase activity.Optionally, said glycopeptide is glycosylated by the enzymes C2GnT andFUT3. Optionally, said glycopeptide is glycosylated by the enzymes C2GnTand FUT7. Optionally, said glycopeptide is glycosylated by the enzymesC2GnT, FUT3 and FUT7. Preferably, said glycopeptide is furtherglycosylated by α(1-3)galactosyltransferase (GT). In a preferredembodiment, said glycopeptide comprises between 1 and 15 of saidrepetitions. Said glycopeptide can be recombinant or purified from abiological fluid. Preferably, said biological fluid is urine, inparticular urine from a subject suffering from a pancreatic pathology,particularly pancreatic cancer. In a particular embodiment, saidglycopeptide is loaded on antigen-presenting cells, preferably dendriticcells.

The invention also relates to the use of a glycopeptide or apharmaceutical or vaccine composition according to the invention asmedicament. In particular, the invention relates to the use of aglycopeptide or a pharmaceutical or vaccine composition according to theinvention for preparing a medicament intended for the preventive orcurative treatment of a disease selected in the group consisting of acancer, an inflammatory disorder, a vascular pathology or an infectionby a pathogen. Preferably, the disease is a breast or pancreas cancer.In a preferred embodiment, the invention relates to the use of aglycopeptide or a pharmaceutical or vaccine composition according to theinvention for preparing a medicament intended for the treatment orprevention of a pancreatic pathology, in particular pancreatic cancer.In a preferred embodiment, the glycopeptide is obtained by purificationfrom the urine of a subject suffering from a pancreatic pathology,preferably pancreatic cancer.

Within an adjuvant formulation, the immunogen, in this case aglycopeptide according to the invention, can be included in particularin a water-in-oil emulsion, by using ISA 51 for example.

A vaccine preparation containing the immunogenic glycopeptide can beadministered in a suitable pharmaceutical formulation to induce animmune response of the systemic type by the intramuscular (im),subcutaneous (sc), intradermal (id) route or of the mucosal type by theintranasal, oral, vaginal or rectal route.

A vaccine preparation containing the immunogenic glycopeptide can alsocontain other immunogens.

A systemic pharmaceutical preparation, administered by the sc, im, idroute, can be a water-in-oil emulsion containing the immunogenicglycopeptide, or an immunogen-embedded calcium phosphate suspension, oraluminium hydroxide adsorbing the immunogen.

The vaccine preparations can be formulated for the intranasal route inthe form of a gel with carbopol as excipient, nose drops or spray andfor the oral route in the form of gastroresistant capsules,gastroresistant granules or sugar coated tablets.

The usual dose, which varies according to the subject and the causalpathology, can be for example from 1 to 100 mg of a glycopeptideaccording to the invention, in particular of the glycopeptide describedin example 4 hereinbelow, administered by the systemic route in humans,every two weeks for 10 to 20 weeks.

The invention also has as object pharmaceutical compositions whichcontain at least one aforementioned glycopeptide, as active ingredient.

In said compositions, the active ingredient is advantageously present atphysiologically effective doses; in particular the aforementionedcompositions contain an effective vaccine dose of at least oneaforementioned active ingredient.

As medicaments, the glycopeptides described hereinabove can beincorporated in pharmaceutical compositions intended for the digestiveor parenteral route.

For example, said pharmaceutical compositions can be solids or liquidsand be supplied as pharmaceutical formulations commonly used in humanmedicine, such as for example simple or sugar-coated tablets, capsules,granules, caramels, suppositories, particularly injectable preparations;they are prepared by the usual methods. The active ingredient(s) can beformulated therein with excipients usually employed in saidpharmaceutical compositions, such as talc, gum arabic, lactose, starch,magnesium stearate, cocoa butter, vehicles which are aqueous or not,animal or vegetable fats, paraffin derivatives, glycols, various wettingagents, dispersives or emulsifiers, preservatives.

The invention also has as object a method of preparation of acomposition such as described hereinabove, characterized in that theactive ingredient(s) are mixed with acceptable excipients, particularlypharmaceutically acceptable, according to established methods.

The invention further has as object the use of a glycopeptide such asdescribed hereinabove, for preparing a medicament intended for thecurative or preventive treatment of pancreatic cancer or breast canceror other pathologies such as a vascular pathology or angiogenesis.

The invention further relates to a method of preventive or curativetreatment of a disease selected in the group consisting of a cancer, aninflammatory disorder, a vascular pathology or an infection by apathogen in a subject, comprising administering an effective dose of aglycopeptide according to the invention.

In a preferred embodiment of cancer treatment, in particular prostatecancer, the glycopeptide can be loaded on an allogeneicantigen-presenting cell. Preferably, the antigen-presenting cell is adendritic cell. In a preferred manner, the antigen-presenting cell isautologous, that is, it has previously been removed from the recipientsubject or it is derived from stem cells originating from the recipientsubject. In an alternative, the antigen-presenting cell originates froma sample taken from an allogeneic subject, that is to say, compatiblewith the recipient. Thus, in this embodiment, the pharmaceutical orvaccine composition according to the invention comprisesantigen-presenting cells loaded or “pulsed” with a glycopeptideaccording to the invention. The invention therefore relates to the useof a pharmaceutical or vaccine composition comprising antigen-presentingcells loaded or “pulsed” with a glycopeptide according to the inventionas medicament, in particular as medicament for the treatment orprevention of breast or pancreatic cancer, preferably pancreatic cancer.In a preferred embodiment, the glycopeptide is obtained by purificationfrom the urine of a subject suffering from a pancreatic pathology,preferably pancreatic cancer. In a most desirable embodiment, theglycopeptide is obtained by purification from the urine of a treatedsubject. Preferably, the subject is human. The invention also relates toa method of curative or preventive treatment of a pancreatic pathology,preferably pancreatic cancer, or breast cancer, comprising: providingthe glycopeptides according to the invention; and administering saidglycopeptides, optionally loaded on antigen-presenting cells, saidadministration allowing to treat, attenuate or prevent a pancreaticpathology, preferably pancreatic cancer, or breast cancer. In apreferred embodiment, the treated disease is pancreatic cancer. Saidglycopeptides can be recombinant or isolated from a biological sample,preferably a biological fluid. In a preferred embodiment, saidglycopeptides are isolated and/or purified from the urine of a subjectsuffering from a pancreatic pathology, preferably pancreatic cancer, orbreast cancer. Preferably, said glycopeptides are isolated and/orpurified from the urine of a treated subject. Thus, the step ofproviding the glycopeptides can comprise the collection of thebiological fluid, preferably urine, and the purification of saidglycopeptides.

The properties of the glycopeptides described hereinabove also justifytheir use in diagnosis, particularly in immunoenzymatic methods.

This is why the invention also has as object a composition for thediagnosis in vitro of the presence of an antibody directed against aglycopeptide described hereinabove, in a human biological sample,characterized in that it contains at least one glycopeptide such asdescribed hereinabove.

The invention also has as object a method for the detection in vitro ofthe presence of induced antibodies in a human subject suffering from anaforementioned pathology, in a human biological sample such as serumand, more particularly, for the diagnosis in vitro of pancreatic canceror a diabetic pathology and originating from the person who is theobject of the diagnosis, characterized in that said biological sample iscontacted with an antigen recognized by an induced antibody in thepatient such as defined hereinabove or hereinbelow, in conditionsallowing the formation of an immunological complex between said antigenand said antibody and in that the immunological complex possibly formedbetween said antibody and antigen is detected.

The invention also has as object the necessary materials or kit for thedetection of induced antibodies in a human subject suffering from anaforementioned pathology, in a biological sample, particularly apossible carrier of said antibodies, characterized in that it comprises:

-   -   at least one glycopeptide such as described hereinabove    -   means for detecting the immunological complex resulting from the        immunological reaction between the antigen and said biological        sample.

The glycopeptides, particularly recombinant, isolated or purified whichare the object of the invention can also be used in the diagnosis ofcertain pathologies such as type I diabetes by detection of circulatingantibodies directed against these structures in patients with suchdiabetic pathology.

In particular, the glycopeptides named C2-F7-16R are used for thediagnosis of type I diabetes.

As the glycopeptides, particularly recombinant, isolated or purifiedwhich are the object of the invention possess very interesting antigenicproperties, they also enable the production of antibodies particularlymonoclonal.

Hence the application also has as object an antibody particularlymonoclonal giving rise to a specific immunological reaction with aglycopeptide such as described hereinabove, with the exception ofantibody J28 and any other antibody meeting the hereinabove definitionand which can be described in the literature. The monoclonal antibodiescan be from any class, in particular IgM, IgD, IgE or IgA. Preferably,the monoclonal antibodies are IgG. An antibody according to theinvention can be modified, regardless of the initial class thereof, toan antibody from another immunoglobulin class, for example by knownmolecular biology methods (Lewis et al., 1992, Hum. AntibodiesHybridomas, 3: 146-52; Lewis et al., 1993, J. Immunol., 151: 2829-38;Hall et al., 1994, Cancer Res., 54: 5178-85; Shepherd & Dean, MonoclonalAntibodies, Oxford University Press, 2000, 479 pages). These samemonoclonal antibodies can be humanized by genetic recombination or byany other method in order to conserve their recognition site for saidrecombinant or natural glycopeptides so as to use them in the conditionsindicated earlier.

In particular an antibody obtained by immunization of a mammal with theaid of a glycopeptide such as described hereinabove, is chosen.

More particularly, the invention relates to the antibodies described inthe examples and in particular the 16D10 antibodies of the IgM typewhich can be produced by the hybridoma deposited with the CollectionNationale de Culture de Microorganismes (CNCM), Institut Pasteur, 28,rue du Dr Roux, 75724 Paris Cédex 15, France on 16 Mar. 2004 under thenumber I-3188 and 8H8 of the IgG type.

Preferably, the invention relates to a monoclonal antibody selected inthe group consisting of the monoclonal antibody 16D10, a fragment orderivative thereof, and an antibody which essentially binds to the sameepitope as monoclonal antibody 16D10. Optionally, said antibody ishumanized, chimeric or human. Preferably, the antibody is of the IgGtype. Optionally, the antibody is a single chain antibody.

An antibody fragment or derivative is understood to mean an entityconserving substantially the same specificity as the antibody inquestion. A fragment thereof can be a Fab, Fab′, F(ab)2 and sFv fragment(Blazar et al., 1997, Journal of Immunology 159: 5821-5833 and Bird etal., 1988, Science 242: 423-426). Fv, Fab, or F(ab)2 fragments accordingto the invention can be obtained by classical enzymatic digestionmethods. A derivative thereof can be a chimeric, humanized or singlechain scFv antibody. Said derivatives can be obtained by classicalgenetic engineering methods. Polynucleotides coding for variable regionsof antibody 16D10 can be obtained for example by cloning said variableregions from a 16D10-antibody-producing hybridoma cDNA library. They canalso be totally or partially prepared by nucleic acid synthesis, basedon the nucleotide sequences of said variable regions. For examplepolynucleotides coding for the CDRs of 16D10 can be synthesized, andincorporated into suitable regions of an other antibody, in particularan antibody of human origin and/or of the IgG type, by known techniquesof CDR grafting, such as those described by Routledge et al. (“Reshapingantibodies for therapy”, in Protein Engineering of Antibody Moleculesfor Prophylactic and Therapeutic Applications in Man, 13-44, AcademicTitles, Nottingham, England (1993)] or by Roguska et al., 1996, ProteinEngineering, 9 (10): 895-904).

The invention also has as object any nucleic acid molecule coding for aprotein comprising the CDRs of antibody 16D10, and any recombinantvector, in particular any expression vector, comprising said nucleicacid molecule, any host cell comprising said nucleic acid or saidvector.

The invention also has as object a method of preparation of ananti-glycopeptide antibody such as described hereinabove, characterizedin that a mammal is immunized with the aid of said glycopeptide or withthe aid of a mixture of FAPP and BSDL isolated from any human or animalbiological tissue or fluid and in particular normal and pathologicalhuman pancreatic juices and the expected antibodies recognizing theC-terminal domain of said proteins are then recovered and purified if sodesired.

The application equally has as object a method of preparation of ananti-glycopeptide antibody such as described hereinabove characterizedin that one clones B lymphocytes originating from a subject immunizedwith the aid of a natural or recombinant glycopeptide such as describedhereinabove or with the aid of a mixture of FAPP and BSDL such asdescribed hereinabove and transformed by Epstein-Barr virus (EBV), thenrecovers the expected antibodies secreted by said transformed Blymphocytes.

In particular, the monoclonal antibodies object of the invention cangive rise to a specific immunological reaction with a glycopeptide suchas described hereinabove.

In a particular embodiment of the invention, the antibody essentiallybinds to the same epitope as the monoclonal antibody 16D10 or J28(respectively produced by hybridoma 16D10 or J28). Preferably, saidantibody is a monoclonal antibody. Preferably, the invention relates tothe monoclonal antibody 16D10 (produced by hybridoma 16D10), but itshall be understood that the invention also relates to monoclonalantibodies which can specifically compete with antibody 16D10. Likewise,in a particular embodiment, the monoclonal antibodies according to theinvention can specifically compete with antibody J28.

The term “essentially binds to the same epitope or determinant as” anantibody of interest means that the antibody “competes” with saidantibody of interest. The term “essentially binds to the same epitope ordeterminant as” an antibody of interest does not mean that the twoantibodies have exactly the same epitope. However, in a particularembodiment, the two antibodies can have the same epitope. The term“essentially binds to the same epitope or determinant as” monoclonalantibody 16D10 means that the antibody “competes” with antibody 16D10.Generally, an antibody which “essentially binds to the same epitope ordeterminant as” an antibody of interest (eg., antibody 16D10) means thatthe antibody “competes” with said antibody of interest for aglycopeptide according to the invention, in particular one or moreproteins selected from BSDL or FAPP or a fragment thereof, preferably aC-terminal part of BSDL or FAPP as described herein, even morepreferably a part comprising one or more repetitions of 11 amino acidsof BSDL or FAPP, or a part or fragment of same. In other examples, anantibody essentially binds to the same epitope or determinant of BSDL orFAPP when the antibody “competes” with the antibody of interest forbinding to BSDL or FAPP.

The term “essentially binds to the same epitope or determinant as” anantibody of interest means that the antibody “competes” with saidantibody of interest for any BSDL and/or FAPP molecule to which saidantibody of interest specifically binds. The term “essentially binds tothe same epitope or determinant as” monoclonal antibody 16D10 means thatthe antibody “competes” with said monoclonal antibody 16D10 for any BSDLand/or FAPP molecule to which said monoclonal antibody 16D10specifically binds. For example, an antibody which essentially binds tothe same epitope or determinant as monoclonal antibodies 16D10 or J28“competes” with said antibodies 16D10 or J28 for binding to BSDL orFAPP, respectively.

The identification of one or more antibodies which essentially bind(s)to the same epitope as the monoclonal antibodies described herein can becarried out by using any immunological screening method in which acompetition between antibodies can be evaluated. Many assays areroutinely carried out and are well known to those skilled in the art(see for example U.S. Pat. No. 5,660,827 granted 26 Aug. 1997, which isspecifically incorporated herein by reference). The determination of theepitope to which an antibody binds is not necessary to identify anantibody which binds, or which essentially binds, to the same epitope asthe monoclonal antibody described herein.

For example, when the candidate antibodies to be studied have beenobtained from different animal sources, or possibly have different Igisotypes, a simple competition assay can be employed wherein the controlantibody (antibody 16D10 for example) and the candidate antibody aremixed (or pre-adsorbed) and contacted with a sample containing eitherBSDL or FAPP, both known to bind to antibody 16D10. Protocols based onELISA, radioimmunological assays, western blot analyses, or the use of aBIACORE analysis (as described, for example, in the Examples) aresuitable for use in simple competition tests.

In particular embodiments, it is possible to first prepare mixtures ofcontrol antibodies (for example, antibody 16D10) with variable amountsof candidate antibodies (for example, approximately 1:10 orapproximately 1:100) before contacting with the BSDL or FAPP antigensample. In another embodiment, the control antibody and the variableamounts of candidate antibodies can simply be mixed at the time ofcontact with the BSDL or FAPP antigen sample. So long as one candistinguish bound antibodies from free antibodies (for example, by usingseparation methods or by washing to remove unbound antibodies) anddistinguish antibody 16D10 from the candidate antibodies (for example,by using secondary antibodies specific of the species or the isotype orby labelling antibody 16D10 with a detectable tag), it will be possibleto determine if the candidate antibodies reduce the binding of antibody16D10 to the BSDL or FAPP antigen, thereby indicating whether thecandidate antibody essentially recognizes the same epitope as antibody16D10. The binding of the control antibody in the presence of acompletely irrelevant antibody can serve as an upper control value. Thelower control value can be obtained by incubating the labelled controlantibody (16D10) with unlabelled antibodies recognizing exactly the sameepitope (16D10), in this way inducing a competition which reduces thebinding of the labelled antibody. In one test, a significant reductionin the reactivity of a labelled antibody in the presence of a candidateantibody indicates that the candidate antibody essentially recognizesthe same epitope, that is to say, it cross-reacts with the labelledantibody (16D10). Any candidate antibody which reduces the binding ofantibody 16D10 to each of the antigens BSDL or FAPP by at leastapproximately 20%, 30%, 40%, 50%, preferably by at least about 60%, oreven more preferably by at least about 70% (for example, 65-100%), at aratio of 16D10 antibody to candidate antibody of between about 1:10 andabout 1:100, is considered to be an antibody which essentially binds tothe same epitope or determinant as antibody 16D10. Preferably, saidcandidate antibodies reduce the binding of antibody 16D10 to the BSDL orFAPP antigen by at least about 90% (for example, about 95%). Preferably,antibody 16D10 also reduces the binding of the candidate antibody to theBSDL or FAPP antigen when the binding is evaluated in the same way,although the degree of reduction in binding may be different.

The competition can be evaluated by a flow cytometry assay for example.In such assay, cells carrying a BSDL or FAPP antigen, for example cellstransfected with BSDL or FAPP, are first incubated with antibody 16D10,for example, then with the candidate antibody labelled with afluorochrome or with biotin. The antibody is considered to compete withantibody 16D10 if the binding observed after preincubation with asaturating amount of 16D10 is about 30%, preferably about 40% about 50%,about 80%, or more (for example, about 90%) of the binding observed (asmeasured by fluorescence) with the antibody without preincubation with16D10. Alternatively, an antibody is considered to compete with antibody16D10 when the binding observed with labelled 16D10 antibody (labelledwith a fluorochrome or biotin) in cells preincubated with a saturatingamount of the candidate antibody is about 80%, preferably about 50%,about 40%, about 30%, or less (for example, about 20%) of the bindingobserved without preincubation with the antibody.

In an advantageous manner, a simple competition assay in which acandidate antibody is preadsorbed and applied at a saturatingconcentration on a surface on which a BSDL or FAPP antigen has beenimmobilized can also be used. The surface in the simple competitionassay is preferably a BIACORE chip (or another support compatible withsurface plasmon resonance analysis). The control antibody (for example,16D10) is then contacted with a surface at a saturating concentration ofBSDL or FAPP and binding of the control antibody to the surface carryingBSDL or FAPP is measured. Said binding of the control antibody iscompared with the binding of the control antibody to the surfacecarrying BSDL or FAPP in the absence of the candidate antibody. In anassay test, a significant reduction in binding of the control antibodyto the surface carrying BSDL or FAPP in the presence of the candidateantibody indicates that the candidate antibody essentially recognizesthe same epitope as the control antibody in such a way that thecandidate antibody cross-reacts with the control antibody. Any candidateantibody which reduces the binding of the control antibody (such asantibody 16D10) to the BSDL or FAPP antigen by at least about 30% orpreferably about 40% can be considered an antibody which essentiallybinds to the same epitope as the control antibody (for example, 16D10).Preferably, said candidate antibody reduces the binding of the controlantibody (for example, 16D10) to the BSDL or FAPP antigen by at leastabout 50% (for example, at least about 60%, at least about 70%, ormore). It shall be understood that the order between the control andcandidate antibodies can be reversed, that is to say, that the controlantibody can be the first to bind to the surface and that the candidateantibody is subsequently contacted with the surface in a competitiontest. Preferably, the antibody displaying the highest affinity for BSDLor FAPP is bound to the surface carrying BSDL or FAPP first, since it ishoped that the reduction in binding observed with the second antibody(presuming that the antibodies cross-react) will be of greateramplitude. Other examples of such tests are described in the examplesand in Saunal and Regenmortel, (1995, J. Immunol. Methods 183: 33-41,the content thereof being incorporated herein by reference).

Said properties are illustrated in the experimental section whichfollows. They justify the use of the monoclonal antibodies describedhereinabove and in particular of the monoclonal antibodies 8H8, 16D10,14H10 and other monoclonal antibodies directed against the C-terminalpart of BSDL or FAPP, for the diagnosis, prognosis and/or prediction ofseveral pancreatic pathologies including pancreatic cancer, pancreatitisand type I diabetes, but also breast cancer or cardiovascular diseases.The diagnostic information can be obtained from serum and/or urineassays.

The antibody 16D10 has particularly interesting properties. In fact, itis specific of pancreatic cancer. It recognizes neither normal tissuesnor other types of tumor tissues. The epitope recognized by antibody16D10 is different from that recognized by antibody J28 because thesetwo antibodies do not compete. These results are described in example 4.

Thus, the invention relates to a method enabling the detection,preferably in vitro, of a pancreatic pathology, in particular pancreaticcancer, comprising contacting a biological sample with an antibodyaccording to the invention and detecting the formation of immunologicalcomplexes resulting from the immunological reaction between saidantibody and said biological sample. Preferably, the antibody is theantibody 16D10, a fragment or derivative thereof, or an antibody whichessentially binds to the same epitope or determinant as the former.Preferably, the biological sample is a sample of pancreatic tissue(biopsy) or a biological fluid. The biological sample can be a tissueslice (immunohistochemistry) or cells contained in the sample or derivedby culturing the sample (immunocytochemistry). The biological fluid canbe selected in the group consisting of serum, urine, pancreatic juicesand milk. Preferably, the biological fluid is urine. The complex can bedetected directly by labelling the antibody according to the inventionor indirectly by adding a molecule which reveals the presence of theantibody according to the invention (secondary antibody,streptavidin/biotin tag, etc.). For example, labelling can beaccomplished by coupling the antibody with radioactive or fluorescenttags. These methods are well known to those skilled in the art.

The invention also relates to the use of an antibody according to theinvention for preparing a diagnostic composition that can be used fordetecting a pancreatic pathology in vivo or in vitro. Preferably, theantibody is the antibody 16D10, a fragment or derivative thereof, or anantibody which essentially binds to the same epitope or determinant asthe former. Preferably, the pancreatic pathology is pancreatic cancer.

In a preferred embodiment, the invention relates to a method enablingthe detection of a pancreatic pathology in a subject, in particularpancreatic cancer, comprising recovering the urine of the subject,contacting said urine with an antibody according to the invention, anddetecting the formation of immunological complexes resulting from theimmunological reaction between said antibody and said urine. Preferably,the antibody is the antibody 16D10, a fragment or derivative thereof, oran antibody which essentially binds to the same epitope or determinantas the former. The antibody can also be the antibody J28, a fragment orderivative thereof, or an antibody which essentially binds to the sameepitope or determinant as the former. Preferably, the pancreaticpathology is pancreatic cancer. The complex can be detected directly bylabelling the antibody according to the invention or indirectly byadding a molecule which reveals the presence of the antibody accordingto the invention (secondary antibody, streptavidin/biotin tag, etc.).These methods are well known to those skilled in the art. Optionally,the method can comprise intermediate steps of treating the urine samplebefore incubation with said antibody. For example, said steps cancomprise concentration of the urine, steps of glycopeptide enrichment orpurification, and the like.

The invention also relates to the use of an antibody according to theinvention for the diagnosis of a pancreatic pathology, in particularpancreatic cancer in a subject. Preferably, the antibody is antibody16D10, a fragment or derivative thereof, or an antibody whichessentially binds to the same epitope or determinant as the former. Theantibody can also be antibody J28 a fragment or derivative thereof, oran antibody which essentially binds to the same epitope or determinantas the former.

The invention relates to a diagnostic kit for a pancreatic pathology, inparticular pancreatic cancer, comprising an antibody according to theinvention. Said kit can additionally comprise means by which to detectthe immunological complex resulting from the immunological reactionbetween the biological sample and said antibody, in particular reagentsenabling the detection of said antibody. In a particular embodiment, thekit comprises the antibody 16D10, a fragment or derivative thereof, oran antibody which essentially binds to the same epitope or determinantas the former.

These properties justify the use of the monoclonal antibodies describedhereinabove for developing new therapeutic protocols for cancer of theexocrine pancreas such as targeting tumor cells with said antibodiescoupled with drugs (chemotherapy) or radioactive elements (radiotherapyand radiodiagnosis) or else with genes modifying the outcome or behaviorof the neoplastic cells (gene therapy).

The antibodies coupled with radioactive elements can be used inradiolocalization (immunoscintigraphy) of primary tumors and metastases(secondary tumors).

Said diagnostic information can be acquired in serum and/or urineassays.

In particular, the invention relates to antibodies according to theinvention, in particular monoclonal, coupled with an antitumoral activesubstance, which can be used in the treatment of pancreatic cancer totarget and destroy pancreatic tumor cells.

Similarly, radioactive elements (or others) can be coupled with saidantibodies and allow a precise localization of primary tumors andmetastases. Said antibodies can also make is possible to clearly andaccurately differentiate normal tissue from tumor tissue.

These same properties justify the use of said monoclonal antibodiesalone, combined or coupled with a diagnostic or therapeutic molecule todevelop new passive immunotherapy protocols for the aforementionedpathologies.

Thus the invention also has as object a composition for the in vitrodiagnosis of a pathology such as described hereinabove, in a humanbiological sample, characterized in that it contains at least onemonoclonal antibody such as described hereinabove, in particular coupledwith at least one radioactive element. Depending on the nature of theradioactive element, said composition can be used curatively toirradiate a region of the pancreas.

As a diagnostic composition, the aforementioned antibodies can be mixedwith acceptable excipients.

The invention also has as object a kit for detecting the presence of aglycopeptide such as described hereinabove appearing in a human subjectafflicted with a diabetic or neoplastic pathology in a human biologicalsample originating from the individual to be diagnosed, characterized inthat it comprises:

-   -   at least one antibody such as described hereinabove,    -   means for detecting the immunological complex resulting from the        immunological reaction between the antibody and the biological        sample.

The preferred conditions of employing the glycopeptides and antibodiesdescribed hereinabove also apply to the other objects of the inventionmentioned earlier, in particular to the diagnostic methods.

The invention also relates to a pharmaceutical composition comprising anantibody according to the invention. Preferably, the antibody is theantibody 16D10, a fragment or derivative thereof, or an antibody whichessentially binds to the same epitope or determinant as the former. Theantibody can also be the antibody J28, a fragment or derivative thereof,or an antibody which essentially binds to the same epitope ordeterminant as the former. The composition can also comprise apharmaceutically acceptable support. In a particular embodiment, saidantibody is coupled with an antitumoral substance.

The invention further relates to the use of an antibody according to theinvention or of a pharmaceutical composition comprising such antibody asmedicament. In particular, the invention relates to the use of anantibody according to the invention or of a pharmaceutical compositioncomprising such antibody for preparing a medicament intended for thepreventive or curative treatment of a pancreatic pathology, preferablypancreatic cancer, or breast cancer. Preferably, the antibody is theantibody 16D10, a fragment or derivative thereof, or an antibody whichessentially binds to the same epitope or determinant as the former. Theantibody can also be the antibody J28, a fragment or derivative thereof,or an antibody which essentially binds to the same epitope ordeterminant as the former. In a particular embodiment, said antibody iscoupled with an antitumoral substance.

The invention relates to a method of preventive or curative treatment ofa subject suffering from a pancreatic pathology, in particularpancreatic cancer, or breast cancer, comprising administering to saidsubject an effective amount of an antibody according to the invention,said administration resulting in a decrease or disappearance of thepancreatic pathology, in particular pancreatic cancer, or breast cancer,in the subject. Preferably, the antibody is the antibody 16D10, afragment or derivative thereof, or an antibody which essentially bindsto the same epitope or determinant as the former. The antibody can alsobe the antibody J28, a fragment or derivative thereof, or an antibodywhich essentially binds to the same epitope or determinant as theformer. In a particular embodiment, said antibody is coupled with anantitumoral substance.

In another protocol the usual dose, which varies according to thesubject being treated and the causal disease, can be for example from 1to 10 mg of the monoclonal antibody described in example 18 hereinbelowper kilogram of body weight administered systemically in humans, once aweek for two weeks.

The invention is also directed at providing new products (glycopeptides,antibodies, etc.) as well as other better, more effective or purerproducts than those described in the prior art.

The invention is illustrated in the following examples.

LEGENDS OF FIGURES

FIG. 1. Study comparing the reactivity of mAbJ28 and mAb16D10 in twopancreatic tumor tissues (PDAC=ADK1 and PDAC4=ADK4).

FIG. 2. Competition test between antibodies mAbJ28 and mAb16D10 forglycopeptide J28.

FIG. 3. Competition test between antibodies mAbJ28 and mAb16D10 in SOJ-6cells.

FIG. 4. Comparative immunodetection analysis in urine from healthysubjects (samples 29 to 36) and subjects with pancreatic cancer (samples1 to 5) with the aid of polyclonal antibody pAbL64, monoclonal antibodymAb8H8 and monoclonal antibody mAbJ28. Urine proteins were separated ona 10% polyacrylamide gel then electrotransferred to a nitrocellulosemembrane. The membranes were incubated with pAbL64, mAb8H8 or mAbJ28.The left lanes (Std) correspond to the molecular weight markers.

EXAMPLES

Preparation of Recombinant Glycopeptides

Recombinant glycopeptides of BSDL (SEQ ID No 9) and FAPP (SEQ ID No 13),were expressed in different strains derived from the well-known cellline CHO-K1, a Chinese hamster ovarian tumor cell line with fibroblastmorphology, which can be obtained in particular from the American TypeCulture Collection ATCC under the number CRL61 (Buck et al., 1958, J.Exp. Med., 108: 945-955).

The CHO-K1 cell line was chosen because it does not show detectableC2GnT glycosyltransferase, α(1 -2)fucosyltransferase, α(1-3)fucosyltransferase, α(1-4) fucosyltransferase or α(1-3)galactosyltransferase activity in standard assay conditions.

Preparation of Plasmids (Cloned cDNA)

The cloned cDNAs enabling the expression of the different glycopeptidecomponents were obtained by reverse transcription, carried out with apolydT polynucleotide probe (18 bases) hybridizing with the polyA tailof messenger RNA extracted from tissue samples of healthy human pancreasor the human pancreatic tumor cell line such as for example the cellline SOJ-6 [Fujii et al., 1990, Hum. Cell., 3: 31-36] originating from ahuman pancreatic adenocarcinoma, according to the method described byChirgwin et al. [1979, Biochemistry, 18: 5294-5299].

Preparation 1: Plasmid pSec-FAPP

The amplification of cDNA coding for fetoacinar pancreatic protein(FAPP) (SEQ ID No 10) was reverse transcribed from 5 μg of total RNAextracted from the human pancreatic tumor line SOJ-6 mentioned earlier(45 minutes at 55° C., reverse transcription kit, Sigma, St Louis, Mo.,USA) and followed by a PCR using the nucleotide primers C-ter/FAPP-BSDLand N-ter/FAPP, defined from the cDNA sequence coding for human BSDL(SEQ ID No 6) in the conditions described hereinbelow. The polymerasechain reaction was carried out with an “Advantage-GC cDNA PCR” kit(Clontech) enabling amplification of nucleotide sequences particularlyrich in guanine and cytosine. The following program was employed: onecycle of 2 minutes at 94° C. followed by 35 cycles of 1 minute at 94° C.(denaturation), 1 minute at 52° C. (primer annealing), and 4 minutes at68° C. (extension), followed by one cycle of 10 minutes at 68° C., in aRobocycler Gradient 96 thermocycler (Stratagene).

The primers had the following sequences:

N-ter/FAPP (SEQ ID No 1): 5′-TTCGTaagcttGCGAAGCTGGGCGCCGTGTACAGAA-3′;C-ter/FAPP-BSDL (SEQ ID No 2):5′-TTTCGTgaattcACGCTAAAACCTAATGACTGCAGCATCTG-3′.

The C-ter/FAPP-BSDL primer (SEQ ID No 2) which was used comprises atermination codon so as to eliminate translation of the c-myc epitope aswell as that of the 6×-histidine tag carried by the commercial vector.

The cDNA (nucleotides 1 to 2169) amplified in this manner (SEQ ID No 12)does not comprise a signal peptide.

The resulting cDNA was then cloned into plasmid pSec-Tag (Invitrogen,Leek, the Netherlands).

Preparation 2: Plasmid pSec-16R

The cDNA coding for the C-terminal part of BSDL (from nucleotide 1089:Phe364 to nucleotide 2169: stop codon; SEQ ID Nos 8 and 9) from RNAextracted from normal pancreas was amplified by PCR using the primersC-ter/FAPP-BSDL (primer SEQ ID No 2, see Preparation 1) and N-ter-Ct.

The N-ter-Ct primer had the following sequence:

N-ter-Ct (SEQ ID No 3): 5′-CGTCTAaagcttTTTGATGTCTACACCGAGTCC-3′.

The polymerase chain reaction was carried out in the conditionsdescribed earlier (see Preparation 1).

The resulting cDNA was then cloned into plasmid pSec-Tag.

Preparation 3: Plasmid pSec-6R

The cDNA coding for the C-terminal part of FAPP (from nucleotide 1089:Phe364 to nucleotide 1839: stop codon; SEQ ID Nos 12 and 13) present inthe plasmid pSec-FAPP was amplified by PCR using nucleotide primers No 2(C-ter/FAPP-BSDL) and No 3 (N-ter-Ct) described hereinabove.

The polymerase chain reaction was carried out in the conditionsdescribed in preparation 1.

The resulting cDNA was then cloned into plasmid pSec-Tag.

Preparation 4: Plasmid pBK-αGT

The cDNA coding for α(1-3) galactosyltransferase (from nucleotide −10 tonucleotide +1122: stop codon) from RNA extracted from mouse heart(Balb/c mice) was amplified by PCR using the primers Cter/αGal andNter/αGal defined from the cDNA sequence coding for mouse α(1-3)galactosyltransferase. These primers had the following sequence:

Nter/α Gal (SEQ ID No 4): 5′-AAAAAgaattcGGAGAAAATAATGAAT-3′. Cter/α Gal(SEQ ID No 5): 5′-AAAAAgggcccACAAAGTCAGACATTAT-3′.

The polymerase chain reaction was carried out with the “Taq PCR MasterMix” kit (Qiagen, Courtaboeuf, France) according to the followingprogram : one cycle of 2 minutes at 94° C. followed by 35 cycles of 1minute at 94° C. (denaturation), 1 minute at 54° C. (primer annealing),and 2 minutes at 74° C. (extension), then one cycle of 10 minutes at 74°C., in a Gene Amp PCR System 2400 thermocycler (Perkin Elmer).

The resulting cDNA was then cloned into plasmid pBK-CMV (Stratagene, LaJolla, Calif., USA).

Preparation 5: Plasmid pcDNA3-C2β6GnT-flag

The cDNA coding for glycosyltransferase Core2 β(1-6)N-acetylglucosaminyltransferase described by Panicot et al. inGlycobiology, 1999, 9: 935-946 was cloned into the vector pCDNA-3 whichat its 3′ end comprises a sequence encoding the flag epitope. Expressionof the flag epitope allows detection of C2GnT expression in thetransformed cells.

The following steps were common to the plasmid preparations:

a) Ligation

The different PCR products were analyzed on a 1% agarose gel, purifiedusing the “Geneclean” kit (Bio 101), then subcloned into the shuttlevector pCR2.1 TOPO and sequenced (Euro Sequences Gènes Service, Paris,France) with the universal M13 and reverse M13 primers. The PCR productswere then released from said shuttle vector by the action of therestriction enzymes HindIII and EcoRI and cloned into the eukaryoteexpression and secretion plasmid pSec-Tag to yield plasmids pSec-FAPPand pSec-16R (16R for C-terminal domain of BSDL) (SEQ ID No 8). Thecomplementary DNA coding for the C-terminal part of FAPP, obtained byPCR, was directly cloned after purification and HindIII/EcoRI digestioninto the pSec-Tag plasmid to yield plasmid pSec-6R (6R for C-terminal ofdomain FAPP) (SEQ ID No 12). Sequencing was carried out with theuniversal T7 primer and the BGH reverse primer synthesized for thispurpose by Euro Sequences Gènes Service.

The PCR product coding for α(1-3) galactosyltransferase was releasedwith the restriction enzymes EcoRI and ApaI, purified, then directlyligated into the EcoRI/ApaI sites of the vector pBK-CMV to yield plasmidpBK-αGT.

b) Bacterial Transformation

A 5 μl aliquot of the ligation product was contacted with 50 μl ofcompetent bacterial cells (Escherichia coli, strain TOP10F′) accordingto the protocol described by Hanahan [Hanahan, 1983, J. Mol. Biol., 166:557-580]. Two microliters of 0.5 M 62 -mercaptoethanol were added andthe sample was kept on ice for 30 minutes, then heat-shocked at 42° C.for 30 seconds, and immediately put back on ice. After 2 minutes, thesample was diluted in 450 μl of SOC medium (Life Technologies). Thebacterial suspension was incubated for 1 hour at 37° C. with shaking.The bacteria were then spread, using glass beads, on Petri dishescontaining Luria-Bertagni agar supplemented with 50 μg/ml ampicillin.

c) Plasmid Purification

The bacterial colonies which appeared after 18 hours of culture onselective agar medium (50 μg/ml ampicillin) were picked and inoculatedin 2 ml of Luria-Bertagni liquid medium containing 50 μg/ml ampicillin.The cultures were incubated for 8 hours at 37° C. with shaking, then 1.5ml of the bacterial suspension was centrifuged at 5,000 g for 5 minutes.The bacterial pellet was taken up in 100 μl of bacterial membranedestabilizing buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, Ribonuclease A100 μg/ml). The bacterial suspension was lysed by addition of 200 μl ofalkaline lysis buffer (200 mM NaOH, 1% SDS) and the pH of thepreparation was neutralized with 150 μl of 3 M potassium acetate pH 5.5.The alkaline lysis and neutralization steps require a 5-minuteincubation on ice. Cell debris, denatured proteins and chromosomal DNAwere eliminated by centrifugation at 10,000 g for 10 minutes at 4° C.The supernatant was then extracted with phenol-chloroform by 1:2dilution in phenol/chloroform/isoamyl alcohol (25/24/1 V/V/V) stabilizedto pH 8 with 100 mM Tris-HCl. The aqueous and organic phases wereseparated by centrifugation at 10,000 g for 2 minutes at roomtemperature. The upper aqueous phase was recovered and diluted 1:3 inabsolute ethanol (−20° C.). Plasmid DNA was precipitated bycentrifugation at 12,000 g for 15 minutes at 4° C., then washed twicewith 70% ethanol. Plasmid DNA was then resuspended in 20 μl ofultra-pure water and stored at −20° C.

Preparation of Cell Clones

Preparation 6: Cell Clone CHO-C2 and General Points Regarding theObtaining of Intermediate Cell Clones

A CHO-K1 cell line was transfected with plasmid pcDNA3-C2β6GnT-flagcomprising the cDNA coding for the glycosyltransferase Core2 β(1-6)N-acetylglucosaminyltransferase (C2GnT), which at its 3′ end comprises asequence coding for the flag epitope. Expression of the flag epitopeallows detection of C2GnT expression in the transformed cells.

To this effect, CHO-K1 cells were grown in HAM-F12 medium. When thecells reached 60 to 80% confluence, the culture medium was removed, thecell surface washed three times with Opti-MEM medium then covered with200 μl of transfection solution diluted in Opti-MEM medium without fetalcalf serum. The transfection solution contained the aforementionedplasmid and a lipofectamine liposome suspension (Life Technologies).

The cells were kept like this for 16 hours at which time thetransfection supernatant was replaced with 2 ml of complete HAM-F12medium containing a suitable selective antibiotic (neomycin, zeocin orhygromycin) for a period of at least four weeks. Clones resistant to theaction of the antibiotic were isolated and then amplified.

The CHO-K1 cell clone selected in this manner with neomycin (250 μg/ml)was named CHO-C2.

Said clone CHO-C2 was then transfected with plasmid pSec-16R asdescribed below for preparation 7. The clone resulting fromtransformation of CHO-C2 cells with plasmid pSEC-16R was selected withzeocin (1 mg/ml) and named CHO-C2-16R.

CHO-K1 cells were also cotransfected with plasmid pCDM7-FUT3 coding forfucosyltransferase 3 described by Kukowska-Lallo et al. (1990, GenesDev. 4: 1288-1303), and plasmid pMamNeo according to the methoddescribed hereinabove. The CHO-F3 clone resulting from transformation ofCHO-K1 cells with plasmid pCDM7-FUT3 was selected on the one hand withneomycin (250 μg/ml) and on the other hand for its high expression ofsialyl Lewis x motifs. Said expression was detected by indirectimmunofluorescence with the aid of the antibody CSLEX-1(Becton-Dickinson). In this manner clone CHO-F3 was obtained.

Said clone CHO-F3 was then transfected with plasmid pSec-16R asdescribed below for preparation 7. The clone resulting fromtransformation of CHO-F3 cells with plasmid pSec-16R and selection withzeocin (1 mg/ml) was named CHO-F3-16R.

CHO-K1 cells were also transfected with plasmid pCDM8-FUT7 coding forfucosyltransferase 7 described by Lowe et al. (1991, J. Biol. Chem. 266:17467-17477) instead of plasmid pCDM7-FUT3, then with plasmid pSec16R.After selection with zeocin and neomycin in the conditions describedearlier, the resulting clone was named CHO-F7-16R.

To prepare the intermediate clones expressing the C-terminal peptide ofFAPP (6R), the aforementioned method was employed by transfecting thecell lines or cell clones CHO-K1, CHO-C2 and CHO-F3 with plasmid pSec-6Rinstead of plasmid pSec16R (see method described in preparation 7).However, only the glycosyltransferases C2GnT and fucosyltransferase 3required to build the J28 glycosylated epitope were used duringpreparation of the cell lines.

Three intermediate cell clones CHO-6R, CHO-C2-6R and CHO-F3-6R were thusobtained after antibiotic selection in the conditions describedhereinabove.

Preparation 7: Cell Clone CHO-16R

CHO-K1 cells were transfected with plasmid pSec-16R, which correspondsto plasmid pSec-Tag (Invitrogen, Leek, the Netherlands) into which thecDNA coding for the C-terminal domain of BSDL was cloned (seepreparation 2). This was carried out as for the preparation of cloneCHO-C2 in preparation 6.

The pSec-Tag plasmid carries a sequence coding for the V-J2C region ofthe mouse immunoglobulin κ light chain. Said sequence directs theprotein encoded by the cDNA cloned in the plasmid to the secretorypathway of the eukaroytic cell in which it has been transfected.

The clone resulting from transformation of CHO-K1 cells with plasmidpSec-16R was named CHO-16R and was selected for expression of thecorresponding (glyco)peptide [(glyco)peptide 16R].

Preparation 8: Cell Clone CHO-C2-F3-16R

Clone CHO-C2 from preparation 6 was transfected with plasmid pCDM7-FUT3and with plasmid pLSVHg (R & D Systems) conferring hygromycinresistance, to yield cell clone CHO-C2-F3.

The CHO-C2-F3 cell line was transfected with plasmid pSec-16R as inpreparation 7. The clone resulting from transformation of CHO-C2-F3cells with plasmid pSec-16R was named CHO-C2-F3-16R.

CHO-C2-F3-16R expresses sialyl Lewis x motifs and shows reactivitytowards antibody pAbL64.

Preparation 9: Cell Clone CHO-C2-F7-16R

This was obtained as in preparation 8, but by using plasmid pCDM8-FUT7instead of plasmid pCDM7-FUT3.

The clone resulting from transformation of CHO-C2-F7 cells with plasmidpSec-16R was named CHO-C2-F7-16R and the corresponding cell line wasdeposited with the Collection Nationale de Culture de Microorganismes(CNCM), Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cédex 15,France on 16 Mar. 2004 under the number I-3189.

The clones CHO-C2-F3-16R and CHO-C2-F7-16R from preparations 8 and 9express sialyl Lewis x motifs and show reactivity towards antibodypAbL64.

Preparation 10: Cell Clone CHO-C2-F3-6R

Clone CHO-C2-F3 from preparation 8 was transfected with plasmid pSec-6R(see preparation 3) as described for preparations 6 and 7 to yield cloneCHO-C2-F3-6R. This clone expresses a peptide bearing among others aglycosylated epitope recognized by monoclonal antibody J28.

Preparation 11: Cell clone CHO-C2-F3-GT-6R

Clone CHO-C2-F3-6R from preparation 10 was transfected with plasmidpBK-αGT (see preparation 4) as described earlier to yield cloneCHO-C2-F3-GT-6R bearing the glycosylated epitope recognized bymonoclonal antibody J28 as well as the glycosylated epitope αGalrecognized by natural circulating antibodies in human blood.

Example 1 Recombinant Glycopeptide Having the Size and theCharacteristics of Those Corresponding to the C-Terminal End of BSDL(CHO-16R)

The recombinant glycopeptide was produced by routine culture of cellclone CHO-16R (preparation 7) for 16 hours at 37° C. in a humid, 5% CO₂atmosphere in Opti-MEM medium (Invitrogen) containing 2 mM L-glutamine,100 U/ml penicillin, 100 mg/ml streptomycin and 0.1% fongizonesupplemented with suitable selective antibiotics. The cells were seededat a density of 2×10⁴ cells/cm².

The culture medium was changed every 48 hours. When the cells reachedconfluence, they were washed in phosphate buffer (PBS) without CaCl₂ andwithout MgCl₂, then detached with trypsin/0.05% EDTA, sedimented by lowspeed centrifugation (400 g for 3 min) and resuspended in 5 ml ofculture medium.

Intracellular immunofluorescence and flow cytometry were used to checkthat the cell clones expressed the recombinant glycopeptide of interest.

The recombinant glycopeptide was purified by liquid chromatographyfollowed by affinity chromatography and analyzed by SDS-PAGE.

It could be seen on the gel that the secreted peptide resolved into twodifferent molecular weight bands of approximately 78 kDa and 83 kDa.

The 83 kDa band probably corresponds to the glycosylated peptide whereasthe 78 kDa band would either be a non-glycosylated form or a form withless glycosylation.

It therefore appears that the aforementioned clone expressed aglycopeptide having the size and the characteristics of thosecorresponding to the C-terminal end of BSDL.

Example 2 Recombinant Glycopeptide Having the Size and theCharacteristics of Those Corresponding to the C-Terminal End of BSDL(CHO-C2-16R)

The method described in example 1 was employed, but by culturing thecell clone CHO-C2-16R (preparation 6).

The recombinant glycopeptide obtained was purified by liquidchromatography followed by affinity chromatography and analyzed bySDS-PAGE.

It could be seen on the gel that the secreted peptide resolved into twodifferent molecular weight bands of approximately 78 kDa and 83 kDa.

It therefore appears that the aforementioned clone expressed aglycopeptide having the size and the characteristics of thosecorresponding to the C-terminal end of BSDL.

Example 3 Recombinant Glycopeptide Having the Size and theCharacteristics of Those Corresponding to the C-Terminal End of BSDL(CHO-F3-16R)

The method described in example 1 was employed, but by culturing thecell clone CHO-F3-16R (preparation 6).

The recombinant glycopeptide obtained was purified by liquidchromatography followed by affinity chromatography and analyzed bySDS-PAGE.

It could be seen on the gel that the secreted peptide resolved into twodifferent molecular weight bands of approximately 78 kDa and 83 kDa.

It therefore appears that the aforementioned clone expressed aglycopeptide having the size and the characteristics of thosecorresponding to the C-terminal end of BSDL.

Example 4 Recombinant Glycopeptide Having the Size and theCharacteristics of Those Corresponding to the C-Terminal End of BSDL(CHO-C2-F3-16R)

The method described in example 1 was employed, but by culturing thecell clone CHO-C2-F3-16R (preparation 8).

The recombinant glycopeptide obtained was purified by liquidchromatography followed by affinity chromatography and analyzed bySDS-PAGE.

It could be seen on the gel that the secreted peptide resolved into twodifferent molecular weight bands of approximately 78 kDa and 83 kDa.

It therefore appears that the aforementioned clone expressed aglycopeptide having the size and the characteristics of thosecorresponding to the C-terminal end of BSDL.

Example 5 Recombinant Glycopeptide Having the Size and theCharacteristics of Those Corresponding to the C-Terminal End of BSDL(CHO-F7-16R)

The method described in example 1 was employed, but by culturing thecell clone CHO-F7-16R (preparation 6).

The recombinant glycopeptide obtained was purified by liquidchromatography followed by affinity chromatography and analyzed bySDS-PAGE.

Only one band of molecular weight 83 kDa was seen on the gel.

Example 6 Recombinant Glycopeptide Having the Size and theCharacteristics of Those Corresponding to the C-Terminal End of BSDL(CHO-C2-F7-16R)

The method described in example 1 was employed, but by culturing thecell clone CHO-C2-F7-16R (preparation 9).

The recombinant glycopeptide obtained was purified by liquidchromatography followed by affinity chromatography and analyzed bySDS-PAGE.

It could be seen on the gel that the secreted peptide resolved into twodifferent molecular weight bands of approximately 78 kDa and 83 kDa.

It therefore appears that the aforementioned clone expressed aglycopeptide having the size and the characteristics of thosecorresponding to the C-terminal end of BSDL.

It therefore appears that all the aforementioned clones (16R) expressedglycopeptides having the size and the characteristics of thosecorresponding to the C-terminal end of BSDL.

Examples 7 to 13 Recombinant Glycopeptides Having the Size and theCharacteristics of Those Corresponding to the C-Terminal End of FAPP

The recombinant polypeptides produced by clones CHO-K1 (control), CHO-6R(Ex 7), CHO-C2-6R (Ex 8), CHO-F3-6R (Ex 9), CHO-C2-F3-6R (Ex 10),CHO-GT-6R (Ex 11), CHO-C2-GT-6R (Ex 12) and CHO-C2-F3-GT-6R (Ex 13),were purified on a polyacrylamide gel or else by liquid and/or affinitychromatography and analyzed by immunodetection using antibody pAbL64described by Abouakil et al., 1988, Biochim. Biophys. Acta, 961:299-308, and mAbJ28. The mouse monoclonal antibody mAbJ28, specific ofthe fucosylated J28 glycopeptide derived from O-glycosylation of theoncofetal form of FAPP, is described by Panicot et al. in Glycobiology,1999, 9: 935-946.

The anti-BSDL polyclonal antibody pAbL64 was obtained by immunizingrabbits after purifying the antigen from human pancreatic juices. Saidantibody perfectly recognizes FAPP, the oncofetal form of BSDL.

The polyclonal antibody specific of the BSDL protein core, namedantipeptide, was obtained by immunizing rabbits with a synthetic peptidecorresponding to the peptide sequence of BSDL comprised between theserine residue in position 346 and glutamine in position 370 coupled tohemocyanin.

Said two antibodies were purified on a protein A-Sepharose 4B column(Pharmacia). The antipeptide was subjected to a second purification stepby affinity chromatography on a column of keyhole limpet hemocyanin(KLH) coupled to Sepharose gel to eliminate anti-KLH antibodies.

The recombinant peptides produced by the different clones of the 6R typewere loaded at 100 μg per lane on a 10% polyacrylamide gel, separated,then electrotransferred to a nitrocellulose membrane. The membranes werethen incubated in the presence of antibody pAbL64 on the one hand, andantibody mAbJ28 on the other hand, and developed.

In both cases, the antibodies employed bound specifically topolypeptides having a size of 50 kDa, corresponding to the expected sizeof the C-terminal glycopeptides of FAPP, except in the case of thenon-transfected CHO-K1 cell line used as negative control.

Examples 14 to 19 Anti-Glycopeptide Monoclonal Antibodies

Recombinant anti-glycopeptide antibodies having the size and thecharacteristics of those corresponding to the C-terminal end of FAPPwere prepared as follows:

Stage A—Immunization of Mice.

Six-week-old male Balb/c mice were immunized according to the followingprotocol:

-   -   Day 0: Intraperitoneal injection of 25 μg of a mixture of FAPP        and BSDL purified from normal and pathological human pancreatic        juices (hereinbelow named BSDL-FAPP antigen) in a 50:50 emulsion        (150 μl NaCl/150 μl complete Freund's adjuvant).    -   Day 15: Intraperitoneal challenge with 25 μg of BSDL-FAPP        antigen in a 50:50 emulsion (150 μl NaCl/150 μl incomplete        Freund's adjuvant).    -   Day 30: Intraperitoneal challenge with 25 μg of BSDL-FAPP        antigen in a 50:50 emulsion (150 μl NaCl/150 μl incomplete        Freund's adjuvant).    -   Day 110: Intraperitoneal challenge with 20 μg of BSDL-FAPP        antigen in a 50:50 emulsion (150 pi NaCl/150 μl incomplete        Freund's adjuvant).    -   Day 140: Intraperitoneal challenge with 20 μg of BSDL-FAPP        antigen in a 50:50 emulsion (150 μl NaCl/150 μl incomplete        Freund's adjuvant).    -   Day 215: Intraperitoneal challenge with 20 μg of BSDL-FAPP        antigen in a 50:50 emulsion (150 μl NaCl/150 μl incomplete        Freund's adjuvant).    -   Day 244: Intravenous injection of 10 μg of BSDL-FAPP antigen in        100 μl of sterile NaCl.    -   Day 247: Cell fusion.

Stage B—Cell Fusion According to the Protocol of Kohler and Milstein.

a) At day 247, the selected mouse was sacrificed and the spleen removedand ground up. The spleen cells were washed in RPMI 1640 medium.P3.X63.Ag8 653 myeloma cells, previously grown in RPMI 1640 mediumcontaining 20% fetal calf serum (FCS), 1% glutamine, 1% nonessentialamino acids and 1% sodium pyruvate were also washed in the same medium.

At the same time, peritoneal macrophages were collected by peritoneallavage of non-immunized Balb/c mice with RPMI.

For hybridoma formation, the spleen cells and myeloma cells were mixedin a tube at a ratio of 5 spleen cells for 1 myeloma cell. Aftercentrifugation, the cell pellet was resuspended in 800 μl of 50%polyethylene glycol 1500 in 75 mM Hepes buffer pH 7.5. After 1 minute ofcontact at 37° C., 20 ml of RPMI 1640 medium were slowly added to thefused cells.

b) The initial culture was carried out on 96-well microtitration platesin the presence of RPMI medium containing 20% fetal calf serum (FCS) andsupplemented with 5×10⁻³ M hypoxanthine, 2×10⁻⁵ M aminopterin and 8×10⁻⁴M thymidine. Next, 5×10³ peritoneal macrophages followed by 1 05 fusedcells were deposited in each well.

Stage C—Cloning and Subcloning.

Each hybridoma selected by the method described in stage D hereinbelowresulted from cloning by a limit dilution technique in which 10, 5, 2, 1and 0.5 cells were statistically distributed into microwells containingperitoneal macrophages. Two subclonings were thus carried out, eachclone and subclone having been replicated then frozen in 90% FCS and 10%dimethylsulfoxide (DMSO). Subclones from the last generation were thenexpanded in vivo to obtain ascites fluid in the Balb/c mice, followed byimmunoglobulin purification on a protein A column.

Stage D—Hybrid Cell Selection Method.

Selection was carried out by a liquid phase ELISA method using culturesupernatant. Five micrograms of BSDL-FAPP antigen in 100 μl ofbicarbonate buffer pH 8.5 were deposited in the wells of a 96-well ELISAplate. The plate was activated for 12 h at 4° C. and saturated for 2hours with 300 μl of 1 mg/ml bovine albumin. The plates were then washedand incubated with 100 μl of cell culture supernatants potentiallycontaining antibodies directed against the BSDL-FAPP antigen. After a2-hour incubation, the plates were washed and incubated for 2 hours withalkaline phosphatase-labelled secondary antibodies. At the end of theincubation, the plates were again washed and incubated withpara-nitrophenylphosphate (100 μl, 1 mg/ml in 0.2 M Tris/HCl buffer pH8.2 and 1 mM CaCl₂). After 1 hour at 37° C., the plates were read on amicroplate reader at 410 nm.

About 15 antibodies were selected for their response in the ELISA test.Additional analyses by Western blot (immuno-imprinting) using FAPP asimmunogenic protein enabled the selection of hybridomas 7B4 (Example14), 11D7 (Example 15), 14H9 (Example 16), 14H10 (Example 17), 16D10(Example 18) and 8H8 (Example 19).

Cells producing the IgM antibody 16D10 were deposited with theCollection Nationale de Culture de Microorganismes (CNCM), InstitutPasteur, 28, rue du Dr Roux, 75724 Paris Cédex 15, France on 16 Mar.2004 under the number I-3188.

Example 20 Preparation of Membrane Glycopeptides of Natural Origin

Membrane glycopeptides carrying epitopes recognized by the monoclonalantibodies described in examples 14 to 19 were prepared in the followingmanner: pancreatic tumor cells were cultivated on a plastic support thendetached therefrom with non-enzymatic dissociation solution (Sigma). Thecell pellet obtained by centrifugation (2 min at 1000 rpm) was sonicated(2×15 sec) and again centrifuged (20 min, 14,000 rpm, 4° C.). The pelletsuspended in phosphate buffer corresponds to membrane glycopeptidescarrying epitopes recognized by the monoclonal antibodies described inexamples 14 to 19.

Example 21

A vaccine having the following composition was prepared:

-   -   membrane glycopeptides from example 20 isolated from HaPT-1        pancreatic cells, 20 μg    -   ALU-gel-ser adjuvant (Serva) 150 μl    -   excipient including water for injections 150 μl

Vaccination was carried out by intraperitoneal injection two weeks thenone week before transplanting the tumor cells into the flank of theanimals. The result of said vaccination was compared to that of aplacebo (injection of isotonic solution).

Example 22

A 0.5 mg/ml injectable isotonic solution of antibodies from example 18(16D10) was prepared.

Control Example 1 Recombinant Glycopeptide Having the Size and theCharacteristics of Those Corresponding to the C-terminal End of BSDL

The method described in example 1 was employed, but by cultivating thecell clone CHO-K1.

The gel did not show any bands at molecular weight 78 kDa or 83 kDa.

PHARMACOLOGICAL STUDY Experiment 1 Use of Recombinant C-TerminalGlycopeptides of BSDL and FAPP in Cellular Immunotherapy of ExocrinePancreatic Cancer

Operating Protocol:

Hamsters (90-100 g) were divided into several groups and vaccinated withHaPT-1 cells inactivated by 30 minutes of UV exposure. The hamsters wereinoculated with the glycopeptides from example 20 and inactivated cellsmixed with ALU-gel-ser adjuvant by intraperitoneal injection once a weekfor two weeks. Two weeks after the last inoculation, the hamstersreceived an ectotopic transplantation of HaPT-1 cells (subcutaneous, onthe flank).

Tumor growth was monitored weekly and tumor volume was compared with thecontrol group vaccinated with a placebo (PBS alone).

Results:

The tumor growth curve was significantly slower in hamsters immunizedwith the glycopeptides from example 20 (antigens represented in largepart by structures recognized by monoclonal antibodies J28 and 16D10(Example 18), directed against the C-terminal domain of BSDL and/orFAPP).

At the end of the experiment, tumor volume in the group vaccinated withinactivated cells was 80% lower than in the control group.

The hamsters were sacrificed and pancreatic and tumor tissue sampleswere removed for immunohistochemical analysis. Monoclonal antibodies J28and 16D10 and polyclonal antibody L64 were used to test for expressionof the 16D10 and J28 epitopes (recognized by monoclonal antibodies J28and 16D10, respectively) and the FAPP protein core.

Examination of the tumor tissue slices revealed a decrease in theexpression of the 16D10 and J28 epitopes (smaller decrease for thelatter), between the group vaccinated with inactivated cells and thecontrol group.

Therefore, the C-terminal glycopeptides of BSDL and FAPP according tothe invention can be used in cellular immunotherapy of exocrinepancreatic cancer.

Experiment 2 Use of the Glycopeptides According to the Invention in theDiagnosis of Diabetic Pathologies

Operating Protocol:

A series of experiments carried out by the ELISA method on sera frompatients suffering from various pancreatic and other pathologies showedthat BSDL, used as antigen to activate the ELISA plates, wasspecifically recognized by antibodies present in approximately 80% ofthe sera from patients with type I diabetes whereas less than 10% of thesera from control patients were reactive. Sera from patients with otherpathologies such as pancreatitis, pancreatic cancer or Graves' diseasewere not reactive (Panicot et al. 1999, Diabetes, 48: 2316-2323).

The recombinant glycopeptides from examples 2, 5 and 6 were tested byWestern blot (or immuno-imprinting).

Results:

BSDL, and in particular the C-terminal domain thereof, was recognized bysera from diabetic patients whereas BSDL was not recognized by sera fromnormal control patients.

The recombinant glycopeptides from examples 2 and 6 were recognized bysera from diabetic (type I) patients.

The control sera (from healthy patients) did not recognize any of saidthree glycopeptides.

Therefore, the natural and recombinant C-terminal glycopeptides of theinvention described in the examples can be used for the diagnosis ofdiabetic pathologies.

Experiment 3 Use of Monoclonal Antibodies Directed Against theC-Terminal Domain of FAPP and/or BSDL in the Diagnosis of PancreaticCancer

Operating Protocol:

Human pancreatic tumor cell lines SOJ-6 and Bx-PC-3 and humannon-pancreatic tumor cell lines Caco-2 and Hep-G2 were treated withmonoclonal antibodies J28, 16D10 (Example 18), and 8H8 (Example 19) andwith polyclonal antibody (pAb) L64 specific of the C-terminal domain ofBSDL and FAPP. The respective antigen-antibody complexes were detectedwith the aid of a fluorescent rabbit or mouse fluorescein conjugatedN-isothiocyanate anti-IgG antibody. FACS analysis was used to detect thepresence of antigens associated with the C-terminal domain of FAPP andBSDL at their surface.

Results:

The monoclonal antibodies J28 and 16D10 (Example 18), and the polyclonalantibody (pAb) L64 specifically recognized the surface of the humanpancreatic tumor cell lines SOJ-6 and Bx-PC-3 and did not recognize thesurface of the human non-pancreatic tumor cell lines which were tested,such as Caco-2 and Hep-G2 cells.

The monoclonal antibodies J28 and 16D10 directed against theglycosylated epitopes expressed specifically on the C-terminal domain ofFAPP and BSDL are therefore capable of distinguishing pancreatic tumorcells from non-pancreatic tumor cells.

Therefore, the monoclonal antibodies directed against the glycosylatedepitopes expressed specifically on the C-terminal domain of FAPP andBSDL can be used in the diagnosis of pancreatic cancer.

In particular said antibodies can determine whether a secondary tumorsite (metastasis) is of pancreatic origin or from a primary tumor ofanother origin.

Experiment 4 Targeting of Pancreatic Neoplastic Cells by the MonoclonalAntibodies of the Invention

A—Study on Human Pancreatic Tissue Sections.

Operating Protocol:

Tissue expression of the glycosylated epitopes recognized by themonoclonal antibodies directed against the C-terminal domain of FAPPand/or BSDL was tested in immunohistochemical studies carried out withthe various monoclonal antibodies of the invention on normal andcancerous human pancreatic tissue sections.

In a first step, pancreatic tissue was obtained from four patients withpancreatic cancer and from five normal pancreas. The tissue sectionswere incubated with the antibodies from examples 18 (16D10) and 19(8H8). In a second step, seven human pancreatic tumor tissue samples andfive non-tumoral tissues (diagnosed by a pathologist) were sliced into5-μm thick sections, dried in acetone at 4° C. and rehydrated inTris/HCl buffer pH 7.6. The sections were then incubated either for 1 hat 25° C. (mAbJ28 and mAb8H8, example 19) or overnight at 4° C.(mAb16D10, example 18), the antibodies being diluted 1:50 in Dakodiluent (EnVision System, Dako, Copenhagen, Denmark). The sections werethen incubated with a suitable alkaline phosphatase-labelled secondaryantibody. The antigen-antibody complex was then detected with thechromogenic substrate Fast-Red. In the case of quantitative analysis byconfocal laser scanning microscopy, the suitable secondary antibody waslabelled with streptavidin-fluoroscein. The sections were then examinedunder a microscope or with a confocal laser fluorescence microscope.

Results:

In the first experiment, the monoclonal antibody from example 18 (16D10)specifically recognized neoplastic cells from four pancreatic tumortissue samples.

No reaction was observed with the five normal pancreatic tissues.

The monoclonal antibody from example 19 (8H8) preferentially recognizednormal pancreatic tissue.

In the second experiment, out of all seven tumor tissue samples studied,seven were recognized by mAb16D10 (example 18) and five by mAbJ28. Theimmunohistochemical results are presented in FIG. 1 and Tables 1 and 2.The mAb16D10 (example 18) showed very high specificity for tumor tissuewhereas mAb8H8 (example 19) only recognized normal pancreatic tissue.mAbJ28 showed intermediate specificity since it preferentiallyrecognized tumor tissue, but also some normal tissue. Table 2 presentsthe confocal laser microscopy results defining the fluorescenceintensity on the antibody-labelled tissue sections. It should be notedthat none of these antibodies recognized the other tumor tissues tested:liver, lung, stomach, colon, esophagus and thyroid.

The monoclonal antibodies of the invention coupled to an antitumoralactive substance can be used in the treatment of pancreatic cancer totarget and destroy pancreatic tumor cells.

Similarly, radioactive (or other) elements can be coupled to saidantibodies and enable a precise localization of primary tumors andmetastases. Said antibodies can also afford a clear and sharpdifferentiation between normal tissue and tumor tissue.

TABLE 1 Semi-quantitative immunohistochemical studies: results obtainedwith antibodies mAb8H8, mAbJ28 and mAb16D10 Label mAb8H8 mAbJ28 mAb16D10Control 1 Acini 4 1 0 Canals 0 0 0 Control 2 Acini 4 1 0 Canals 0 0 0Control 3 Acini 4 1 0 Canals 0 0 0 Control 4 Acini 4 1 0 Canals 0 0 0Control 5 Acini 4 1 0 Canals 0 0 0 PDAC1 0 0 4 PDAC2 0 3 4 PDAC3 0 2 4PDAC4 0 0 4 PDAC5 1 3 4 PDAC6 0 2 4 PDAC7* 0 1 3 CCA* 0 0 0 ECA* 0 0 0LiCa* 0 0 0 LuCa 0 0 0 SCA* 0 0 0 TCA* 0 0 0 C, control; PDAC,pancreatic adenocarcinoma; LuCA, lung carcinoma; CCA, colon carcinoma;ECA, esophageal carcinoma; LiCa, liver carcinoma; SCA, stomachcarcinoma; TCA, thyroid carcinoma. Tissues were obtained from theBioChain Institute (USA). The number of labelled cells is expressedsemi-quantitatively as follows: 0, no labelling; 1: from 1 to 10% ofcells were labelled; 2: from 10 to 30% of cells were labelled; 3: from30 to 40% of cells were labelled and 4: >40% of cells were labelled.

TABLE 2 Fluorescence intensity on sections labelled with the antibodiesof the invention. Fluorescence intensity Case mAb8H8 mAbJ28 mAb16D10Control 1 92.33 ± 2.80 19.11 ± 8.75  31.41 ± 1.19 Control 2 138.72 ±3.36  14.54 ± 6.57  8.16 ± 5.16 Control 3 110.95 ± 2.71  17.41 ± 8.09 0Control 4 109.04 ± 4.58   26.49 ± 12.01 0 Control 5 114.14 ± 1.26  35.56 ± 16.30 0 PDAC1 0 53.12 ± 2.12 124.02 ± 2.43 PDAC 2 36.47 ± 0.3587.88 ± 4.28 129.84 ± 6.35 PDAC 3 0 89.60 ± 2.84 123.27 ± 6.16 PDAC 417.06 ± 7.64 50.61 ± 2.36 143.95 ± 2.71 PDAC 5 40.42 ± 0.22 88.92 ± 5.48135.06 ± 4.03 PDAC 6 0 59.97 ± 1.64 129.30 ± 1.91 PDAC 7* 18.65 ± 8.3872.74 ± 4.84 132.34 ± 2.75 C, control; PDAC, pancreatic adenocarcinoma.Tissues were obtained from the BioChain Institute (USA). In each casethe mean ± standard error of labelling on six different regions of eachtissue slice are shown. Significant differences were observed betweencontrol and pancreatic tumor tissue for mAb16D10 (P < 0.001), mAb8H8 (P< 0.001) and mABJ28 (P < 0.05).

B—ELISA Competition Studies Between mAbJ28 and mAb16D10 (Example 18) onthe Recombinant Glycopeptide Carrying the J28 Glycotope.

Operating Protocol:

The glycosyltransferases involved in formation of the oncofetalglycotype J28 were characterized and said glycotope was reproduced exvivo by genetic engineering. It was reconstructed in CHO cells from therecombinant C-terminal peptide of FAPP (composed of 6 repeatedsequences) and two glycosyltransferases, α(1-3/4) fucosyltransferaseFUT3 and Core 2 β(1-6) N-acetylglucosaminyltransferase required to buildsaid structure. The recombinant C-terminal glycopeptide of FAPP carryingthe J28 glycotype (example 10) was secreted in the culture supernatantof this cell line.

Competition experiments were carried out with an ELISA test in thepresence of culture supernatant containing the recombinant C-terminalglycopeptide of FAPP carrying the J28 glycotype (example 10),biotinylated mAbJ28 and increasing concentrations of mAb16D10 (example18) as competitor with the aim of demonstrating the specificity of saidantibodies.

The principle of the protocol was as follows:

1. The bottom of the wells was coated with concentrated culturesupernatant containing the recombinant J28 glycopeptide diluted 1:10,1:20, 1:50, 1:100.

2. Biotinylated mAbJ28 was added at 10 ng per well (Exp. 1) or 5 ng perwell (Exp. 2) together with increasing concentrations of purifiedmAb16D10.

3. Biotinylated mAbJ28 was recognized with the aid of an alkalinephosphatase-conjugated anti-biotin antibody (anti-biotin Ab control onmAb26D10=0).

Results:

FIG. 2 presenting the results of two experiments shows that there was nosignificant loss in the reactivity of mAbJ28 for its glycotope coated atthe bottom of the wells in the presence of increasing concentrations ofmAb16D10 (example 18) used as competitor (even with a four-fold excess).

C—FACS Competition Study Between Antibodies mAbJ28 and mAb16D10 on humanpancreatic tumor cell line SOJ-6.

Operating Protocol:

The monoclonal antibodies mAbJ28 and mAb16D10 specifically recognizedhuman pancreatic tumor cell lines SOJ-6, Bx-PC-3 and did not recognizethe human non-pancreatic tumor cell lines tested.

Preliminary competition experiments by FACS were carried out on thehuman pancreatic tumor cell line SOJ-6 in the presence of monoclonalantibody mAbJ28 coupled to a fluorochrome (Alexa 488) and increasingconcentrations of monoclonal antibody mAb16D10 (example 18) used ascompetitor in the form of ascites fluid.

The principal of the protocol was as follows:

1. Human pancreatic tumor SOJ-6 cells were detached, fixed and saturatedwith BSA at 4° C.

2. Fluorescent-labelled monoclonal antibody mAbJ28 was added at 2 ng persample in the presence of increasing concentrations of ascites fluidcontaining mAb16D10 at 1:100, 1:40, 1:20, 1:20, 1:10, 1:4 and 1:2dilution.

3—The cells were analyzed by FACS.

Results:

The results are shown in FIG. 3. Despite a very low labelling of thecells (due to the first experiments with Alexa-488-labelled mAbJ28), nosignificant loss of reactivity of antibody mAbJ28 for its glycotype wasobserved in the presence of increasing concentrations of antibodymAb16D10 used as competitor.

Experiment 5 Use of the Monoclonal Antibodies of the Invention in theTreatment of Exocrine Pancreatic Cancer

Experimental Protocol:

Two groups of six-week-old nude mice (NMRI mice) received anintraperitoneal inoculation on Day −1 either with monoclonal antibody16D10 from example 18 at a dose of 150 μl of a 0.5 mg/mg isotonicantibody solution or with an isotonic placebo solution.

At Day 0, 2×10⁶ SOJ-6 human pancreatic tumor cells were subcutaneouslyinjected into the right flank. The animals were then inoculated withmonoclonal antibody 16D10 or isotonic solution at Days +1, +3, +6, +8and +10 (in the quantities indicated hereinabove).

The volume of the palpable subcutaneous tumors was measured in eachanimal at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

Results:

The tumor growth rate in animals treated with the monoclonal antibodyfrom example 18 was 10 times lower than that of placebo-treated animals.

At the end of the experiment (10 weeks post-inoculation), the medianvolume of tumors removed from control animals was 1.25 versus a markedlylower median value of 0.15 cm³ in the treated animals.

The monoclonal antibodies directed against the C-terminal domain of FAPPand/or BSDL and in particular the monoclonal antibody from example 18can therefore be used in therapy for pancreatic cancer.

Experiment 6 Use of the Monoclonal Antibodies Directed Against theC-terminal Domain of FAPP and/or BSDL in the Urinary Diagnosis ofPancreatic Cancer

Operating Protocol:

A series of experiments carried out by Western blot, liquidchromatography and mass spectrometric characterization led to theidentification of intact BSDL in the urine of healthy patients. In lightof these findings, the different glycosylated epitopes J28, 16D10carried by the C-terminal domain of BSDL/FAPP and which appear inpancreatic adenocarcinoma, might also be present in the urine ofpatients suffering from this cancer.

Results:

FIG. 4 showing the initial results obtained in a preliminary Westernblot experiment reveals the presence of the glycosylated J28 epitopecarried by BSDL/FAPP in urine samples from patients with pancreaticadenocarcinoma, and the absence thereof in urine samples from healthyindividuals.

FIG. 4 shows that both healthy subjects and cancer patients had one ortwo bands displaying immunoreactivity towards pAbL64 and mAb8H8 (example19) located at 110 kDa. On the other hand, the two groups displayeddifferent reactivity towards mAbJ28, a mAbJ28-immunoreactive band at 110kDa being seen in cancer patients but not in healthy subjects.

Experiment 7 Use of Urinary C-terminal Glycopeptides of BSDL and FAPP inCellular Immunotherapy of Exocrine Pancreatic Cancer

Operating Protocol:

Preliminary results showed that the glycosylated J28 epitope carried byBSDL/FAPP was present in urine samples from patients with pancreaticadenocarcinoma tested at the present time whereas it was absent in urinesamples from healthy individuals. The presence of said tumor marker inurine, which can be obtained in large quantities without the use ofinvasive methods, can serve as a source of antigen in order to developdifferent active immunotherapy protocols such as described by Ramanathanet al., Cancer Immunol Immunoth, 2004, 54: 254-64; O'Neill et al.,Blood, 2004,104: 2235-46; Vlad et al., J Exp Med, 2002, 196: 1435-46;Schmidt et al., Cancer Res, 2003, 63: 8962-67. One such avenue is todevelop autologous cell immunotherapy protocols (as described byYamanaka et al., Br J Cancer, 2003, 89: 1172-9; Svane et al., CancerImmunol Immunother, 2004, 53: 633-41; Yu et al., Cancer Res, 2004, 64:4973-9) using dendritic cells from patients who have their pancreatictumor resected. The patient's dendritic cells are then loaded ex vivowith his own antigens, that is to say, with the C-terminalglycopeptide(s) of BSDL/FAPP carrying the J28 glycotope(s) (and perhapsalso the 16D10 glycotope) obtained from the urine, before beingreinjected in the patient to induce an immune response directed againstresidual tumor foci which express said same antigens.

Said strategy of human dendritic cell activation, currently underevaluation using as tumor antigen the recombinant C-terminalglycopeptide of FAPP carrying the glycosylated J28 epitope (example 10),can also be carried out by using the urinary C-terminal glycopeptide ofBSDL/FAPP carrying said glycosylated epitopes. The purificationprotocols (different steps of centrifugation, ultrafiltration andconcentration, combined with liquid chromatography on exclusion andaffinity columns) developed in order to purify and characterize BSDL inthe urine of healthy individuals are used to purify BSDL/FAPP carryingthe J28 glycotope (and also that of 16D10) from urine samples ofpatients with pancreatic adenocarcinoma. The protocol is as follows:Native urine is centrifuged to eliminate cellular debris and partiallydesalted by ultrafiltration on a Centripreps® YM-10, then completelydesalted on a Sephadex® G-25 column. The recovered fractions arelyophilized, then taken up in 10 mM Tris-HCl elution buffer, pH 7.8, 400mM NaCl, and loaded on Sephacryl™ S-200 gel to separate the proteinsaccording to their molecular mass. Fractions containing BSDL/FAPPcarrying the J28 glycotope (and/or the 16D10 glycotype) are collected,dialyzed and lyophilized. The lyophilizates obtained after molecularsieving are rehydrated in PBS, and BSDL/FAPP carrying the J28 glycotope(and perhaps also the 16D10 glycotope) is purified either byimmunoaffinity chromatography on an agarose-mAbJ28 column or on aheparin Sepharose column. Purified BSDL/FAPP carrying the J28 glycotope(and/or the 16D10 glycotype) is digested with trypsin or cyanogenbromide to obtain the C-terminal glycopeptide of BSDL/FAPP carrying saidglycotopes, as described elsewhere to isolate the C-terminal domain ofBSDL/FAPP (Mas et al., 1997, Glycobiology, 7: 745-752). The dendriticcells of patients with pancreatic adenocarcinoma are cultured in thepresence of said glycopeptide carrying the J28 glycotope (and/or the16D10 glycotype) purified from their own urine. The presence of theglycopeptides at the surface of the pulsed mature dendritic cells willbe checked by confocal microscopy before the cells are reinjected in thepatient to induce an anti-tumor response.

1. A method of detection in vitro of a subject suffering from apancreatic pathology, comprising contacting a biological sample from thesubject with an antibody and detecting the formation of immunologicalcomplexes resulting from the immunological reaction between saidantibody and said biological sample; wherein said antibody canspecifically recognize a glycopeptide comprising 1 to 40 repetitions ofthe peptide sequence described in SEQ ID NO: 14 and glycosylated by oneor more enzymes having ose-transferase activity selected in the groupconsisting of Core 2 β(1-6) N-acetylglucosaminyltransferase (C2GnT),fucosyltransferase FUT3 which has α(1-3) and α(1-4) fucosyltransferaseactivity, or fucosyltransferase FUT7 which has α(1-3) fucosyltransferaseactivity.
 2. The method according to claim 1, wherein said antibody isthe antibody J28, an antigen binding fragment or derivative thereof, oran antibody which essentially binds to the same epitope or determinantas the J28 antibody.
 3. The method according to claim 1, wherein saidantibody is the antibody 16D10, an antigen binding fragment orderivative thereof, or an antibody which essentially binds to the sameepitope or determinant as the 16D10.
 4. The method according to claim 1,wherein said biological sample is a sample of pancreatic tissue.
 5. Themethod according to claim 1, wherein said biological sample is abiological fluid selected from pancreatic juices, serum or urine.
 6. Themethod according to claim 1, wherein the method enables the detection ofa subject suffering from pancreatic cancer.
 7. A composition of mattercomprising: a) pharmaceutical or vaccine composition comprising: i) aglycopeptide comprising 1 to 40 repetitions of the peptide sequencedescribed in SEQ ID NO: 14 and glycosylated by one or more enzymeshaving ose-transferase activity selected in the group consisting of Core2 β(1-6) N-acetylglucosaminyltransferase (C2GnT), fucosyltransferaseFUT3 which has α(1-3) and α(1-4) fucosyltransferase activity, orfucosyltransferase FUT7 which has α(1-3) fucosyltransferase activity;ii) a glycopeptide comprising 1 to 40 repetitions of the peptidesequence described in SEQ ID NO: 14 and glycosylated by the enzymesC2GnT and FUT3; iii) a glycopeptide comprising 1 to 40 repetitions ofthe peptide sequence described in SEQ ID NO: 14 and glycosylated by theenzymes C2GnT and FUT7; or iv) a glycopeptide comprising 1 to 40repetitions of the peptide sequence described in SEQ ID NO: 14 andglycosylated by the enzymes C2GnT, FUT3 and FUT7; or b) a pharmaceuticalcomposition comprising an antibody that can specifically recognize aglycopeptide comprising 1 to 40 repetitions of the peptide sequencedescribed in SEQ ID NO: 14 and glycosylated by one or more enzymeshaving ose-transferase activity selected in the group consisting of Core2 β(1-6) N-acetylglucosaminyltransferase (C2GnT), fucosyltransferaseFUT3 which has α(1-3) and α(1-4) fucosyltransferase activity, orfucosyltransferase FUT7 which has α(1-3) fucosyltransferase activity. 8.The composition of matter according to claim 7, wherein said compositionof matter comprises a pharmaceutical composition comprising an antibodyand: i) the glycopeptide specifically recognized by said antibody isglycosylated by the enzymes C2GnT and FUT3; ii) the glycopeptidespecifically recognized by said antibody is glycosylated by the enzymesC2GnT and FUT7; iii) the glycopeptide specifically recognized by saidantibody is glycosylated by the enzymes C2GnT, FUT3 and FUT7; iv) theglycopeptide specifically recognized by said antibody comprises between1 and 15 of said repetitions; v) said antibody is the antibody J28, anantigen binding fragment or derivative thereof, or an antibody whichessentially binds to the same epitope or determinant as the J28antibody; or vi) said antibody is the antibody 16D10, an antigen bindingfragment or derivative thereof, or an antibody which essentially bindsto the same epitope or determinant as the 16D10 antibody.
 9. Thecomposition of matter according to claim 7, wherein said pharmaceuticalor vaccine composition comprises a glycopeptide and said glycopeptide isfurther glycosylated by α(1-3)galactosyltransferase (GT).
 10. Thecomposition of matter according to claim 7, wherein said pharmaceuticalor vaccine composition comprises a glycopeptide and said glycopeptidecomprises between I and 15 of said repetitions.
 11. The composition ofmatter according to claim 7, wherein said pharmaceutical or vaccinecomposition comprises a glycopeptide and said glycopeptide isrecombinant.
 12. The composition of matter according to claim 7, whereinsaid pharmaceutical or vaccine composition comprises a glycopeptide andsaid glycopeptide purified from a biological fluid.
 13. The compositionof matter according to claim 7, wherein said pharmaceutical or vaccinecomposition comprises a glycopeptide and said glycopeptide is loaded onantigen-presenting cells.
 14. A method for a the treatment of apancreatic disease in a subject comprising administering an effectivedose of a pharmaceutical composition comprising a glycopeptidecomprising 1 to 40 repetitions of the peptide sequence described in SEQID NO: 14 and glycosylated by one or more enzymes having ose-transferaseactivity selected in the group consisting of Core 2 β(1-6)N-acetylglucosaminyltransferase (C2GnT), fucosyltransferase FUT3 whichhas α(1-3) and α(1-4) fucosyltransferase activity, or fucosyltransferaseFUT7 which has α(1-3) fucosyltransferase activity.
 15. The methodaccording to claim 14, wherein the pancreatic disease is pancreaticcancer.
 16. A method for the treatment of a pancreatic disease in asubject comprising administering an effective dose of a pharmaceuticalcomposition comprising an antibody that can specifically recognize aglycopeptide comprising 1 to 40 repetitions of the peptide sequencedescribed in SEQ ID NO: 14 and glycosylated by one or more enzymeshaving ose-transferase activity selected in the group consisting of Core2 β(1-6) N-acetylglucosaminyltransferase (C2GnT), fucosyltransferaseFUT3 which has α(1-3) and α(1-4) fucosyltransferase activity, orfucosyltransferase FUT7 which has α(1-3) fucosyltransferase activity.17. The method according to claim 16, wherein the pancreatic disease ispancreatic cancer.